Compositions, methods and systems for polymerase chain reaction assays

ABSTRACT

Methods, devices, systems and compositions for detecting nucleic acids in polymerase chain reaction assays, such as droplet digital polymerase chain reaction (ddPCR) assays, using intercalating dyes. A dual surfactant system with at least one fluorosurfactant and at least one non-ionic non-fluorosurfactant may be employed for droplet generation and nucleic acid detection.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/201,752, filed Mar. 7, 2014, now U.S. Pat. No. 9,822,393. The '752application claims the benefit of U.S. Provisional Application No.61/775,415, filed Mar. 8, 2013. Each of these priority applications isincorporated herein by reference in its entirety for all purposes.

BACKGROUND

An assay is an investigative procedure for determining the presence,quantity, activity, and/or other properties or characteristics ofcomponents in a sample. Very often, the components of interest within asample e.g., a nucleic acid, an enzyme, a virus, a bacterium are onlyminor constituents of the sample and may, therefore, be difficult todetect or quantify.

An example of a biological assay is a polymerase chain reaction (PCR)assay. Certain types of PCR can be quantitative in specific settings.For example, real-time PCR (which generally involves monitoring theprogression of amplification using fluorescence probes) may permitquantification of target nucleic acids in a sample, particularly wherethe target nucleic acids are somewhat abundant.

Droplet digital polymerase chain reaction (ddPCR) is a popular tool forquantitative measurement of absolute DNA concentration. As opposed tothe relative measurement obtained from the real-time PCR reactions,ddPCR enables absolute measurement of target deoxyribonucleic acid(DNA), ribonucleic acid (RNA) molecules. Absolute quantification isadvantageous in several applications, such as measuring copy numbervariation, detecting rare sequences and mutations, and analyzing geneexpression reaction.

SUMMARY

In an aspect, the present disclosure provides a method of generating aplurality of droplets. The method comprises providing an oil compositioncomprising an oil and a fluorosurfactant, providing an aqueouscomposition comprising a target nucleic acid, a non-ionicnon-fluorosurfactant and an intercalating dye, and contacting the oilcomposition of with the aqueous composition, thereby generating aplurality of droplets suspended in a continuous phase. Thefluorosurfactant can be selected from the group consisting of

wherein each of A and B is oxygen (O) or NR₁, wherein R₁ is H or analkyl group; each of m₁ and m₂ is a number from about 10 to 100; n is anumber from about 10 to 60;

wherein m₃ is a number from about 20 to 50; n₂ is a number from about 8to 30; and R is H or an alkyl group; and

wherein m₄ is a number from about 20 to 50; each of n₃ and n₄ is anumber from about 5 to 25; n₅ is a number from about 0 to 3; and each ofR₁ and R₂ is an alkyl group or H. In some cases, the fluorosurfactantcan be non-ionic. In some cases, the fluorosurfactant can comprise amixture of Formula I, Formula II, and/or Formula III. For example, thefluorosurfactant can comprise a mixture of Formula I and Formula II. Insome examples, the mixture can further comprise Formula XI:

wherein n is a number from about 10 to 100.

In some cases, the fluorosurfactant can have a purity of at least about90% (w/w). For example, the fluorosurfactant can comprise at least about90% (w/w) of a mixture of Formula I and Formula II. Further, the mixtureof Formula I and Formula II can be in a ratio of at least about 90:10(w/w). In some cases, the fluorosurfactant mixture can comprise about0.5% to 5% (w/w) of Formula XI. In certain cases, the oil formulationcan comprise less than about 0.5% (w/w) of Formula XI.

In some cases, the method can further comprise thermally cycling theplurality of droplets to amplify the target nucleic acid and detectingthe target nucleic acid. In some cases, the fluorosurfactant can be ofFormula I. Each of A and B can be 0. Each of m₁ and m₂ can be from about20 to 40, and n can be from about 15 to 30. For example, each of m₁ andm₂ can be about 35 and n can be about 22. In certain cases, thefluorosurfactant can comprise a mixture of Formula I and Formula II. Insome cases, the mixture can further comprise Formula XI. Thefluorosurfactant can have a hydrophilic-lipophilic balance of at leastabout 0.5. The fluorosurfactant can have a hydrophilic-lipophilicbalance of less than about 5.0. The fluorosurfactant can have ahydrophilic-lipophilic balance in a range from about 0.5 to 5.0. Forexample, the fluorosurfactant can have a hydrophilic-lipophilic balanceof about 1.5.

The non-ionic non-fluorosurfactant can be a copolymer of ethylene oxideand propylene oxide. The non-ionic non-fluorosurfactant can be atriblock copolymer of polyethylene oxide-polypropyleneoxide-polyethylene oxide. In some examples, the non-ionicnon-fluorosurfactant can be Pluronic®. For example, the non-ionicnon-fluorosurfactant can be Pluronic® F-98. The concentration of thePluronic® F-98 can be in a range from about 0.1%-3% (weight percent).

The oil can comprise a fluorous oil. The fluorous oil can be2-trifluoromethyl-3-ethoxydodecafluorohexane (HFE-7500) or1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)pentane(HFE-7300). The intercalating dye can be EvaGreen® or SYBR® Green. Thecontinuous phase can comprise the oil.

The plurality of droplets can be aqueous droplets encapsulated by theoil. In some cases, less than about 5% of the intercalating dye can bedetected outside of the aqueous phase of the aqueous droplets afterabout 5 thermal cycles. In some cases, less than about 5% of theintercalating dye can be detected outside of the aqueous phase of theaqueous droplets after about 10 thermal cycles. In some cases, less thanabout 5% of the intercalating dye can be detected outside of the aqueousphase of the aqueous droplets after about 20 thermal cycles. In somecases less than about 5% of the intercalating dye can be detectedoutside of the aqueous phase of the aqueous droplets after about 50thermal cycles. In some cases, less than about 5% of the intercalatingdye can be detected outside of the aqueous phase of the aqueous dropletsafter about 100 thermal cycles.

In some cases, less than about 10% of the intercalating dye can bedetected outside of the aqueous phase of the aqueous droplets afterabout 5 thermal cycles. In some cases, less than about 10% of theintercalating dye can be detected outside of the aqueous phase of theaqueous droplets after about 10 thermal cycles. In some cases, less thanabout 10% of the intercalating dye can be detected outside of theaqueous phase of the aqueous droplets after about 20 thermal cycles. Insome cases, less than about 10% of the intercalating dye can be detectedoutside of the aqueous phase of the aqueous droplets after about 50thermal cycles. In some cases, less than about 10% of the intercalatingdye can be detected outside of the aqueous phase of the aqueous dropletsafter about 100 thermal cycles.

On average, each of the plurality of droplets can comprise less than5.5×10¹² target nucleic acids after thermal cycling. The detecting cancomprise measuring fluorescence from the intercalating dyes.

Another aspect of the present disclosure provides a droplet generator,comprising a first channel in fluid communication with a carrier fluidsource and a second channel in fluid communication with a sample source.The first channel meets the second channel at an intersection. A dropletchannel is in fluid communication with the intersection. During use, anemulsion comprising one or more droplets generated at the intersectionflows along the droplet channel. The carrier fluid source comprises anoil and a fluorosurfactant. The fluorosurfactant can be selected fromthe group consisting of:

wherein each of A and B is oxygen (O) or NR₁, wherein R₁ is H or analkyl group; each of m₁ and m₂ is a number from about 10 to 100; n is anumber from about 10 to 60;

wherein m₃ is a number from about 20 to 50; n₂ is a number from about 8to 30; R is H or an alkyl group; and

wherein m₄ is a number from about 20 to 50; each of n₃ and n₄ is anumber from about 5 to 25; n₅ is a number from about 0 to 3; and each ofR₁ and R₂ is an alkyl group or H. The sample source can comprise atarget nucleic acid, a non-ionic non-fluorosurfactant and anintercalating dye. In some cases, the fluorosurfactant can be non-ionic.In some cases, the fluorosurfactant can be a mixture of Formula I,Formula II, and/or Formula III. For example, the fluorosurfactant can bea mixture of Formula I and Formula II. In some examples, the mixture canfurther comprise Formula XI:

wherein n is a number from about 10 to 100.

In some cases, the fluorosurfactant can have a purity of at least about90% (w/w). For example, the fluorosurfactant can comprise at least 90%(w/w) of a mixture of Formula I and Formula II. Further, the mixture ofFormula I and Formula II can be in a ratio of at least about 90:10(w/w). In some cases, fluorosurfactant mixture can comprise about 0.5%to 5% (w/w) of Formula XI. In certain cases, the oil formulation cancomprise less than about 0.5% (w/w) of Formula XI.

In another aspect, the present disclosure provides a composition,comprising a fluorosurfactant, a non-ionic non-fluorosurfactant and anintercalating dye. The fluorosurfactant can be selected from the groupconsisting of:

wherein each of A and B is oxygen (O) or NR₁, wherein R₁ is H or analkyl group; each of m₁ and m₂ is a number from about 10 to 100; n is anumber from about 10 to 60;

wherein m₃ is a number from about 20 to 50; n₂ is a number from about 8to 30; R is H or an alkyl group; and

wherein m₄ is a number from about 20 to 50, each of n₃ and n₄ is anumber from about 5 to 25, and n₅ is a number from about 0 to 3; andeach of R₁ and R₂ is H or an alkyl group. In some cases, thefluorosurfactant can be non-ionic. In some cases, the fluorosurfactantcan be a mixture of Formula I, Formula II, and/or Formula III. Forexample, the fluorosurfactant can be a mixture of Formula I and FormulaII. In some examples, the mixture can further comprise Formula XI. Insome cases, the fluorosurfactant can have a purity of at least about 90%(w/w). For example, the fluorosurfactant can comprise at least 90% (w/w)of a mixture of Formula I and Formula II. Further, the mixture ofFormula I and Formula II can be in a ratio of at least about 90:10(w/w). In some cases, fluorosurfactant mixture can comprise about 0.5%to 5% (w/w) of Formula XI. In certain cases, the oil formulation cancomprise less than about 0.5% (w/w) of Formula XI.

In another aspect, the present disclosure provides a copolymer ofFormula I wherein each of A and B is oxygen (O) or NR₁, wherein R₁ is Hor an alkyl group, each of m₁ and m₂ is a number from about 10 to 100,and n is a number from about 10 to 60. In some cases, the copolymer canhave a purity of at least about 90% (w/w).

In another aspect, the present disclosure provides a method ofsynthesizing a copolymer. The method comprises heating a solution ofperfluoropolyether carboxylic acid in a first fluorous oil in thepresence of oxalyl chloride and a catalytic amount of dimethylformate(DMF) to form an acid chloride. Next, the acid chloride can be reactedwith polyethylene glycol in a solvent comprising a second fluorous oiland an ether in the presence of an amine to form a product mixturecomprising a perfluoropolyether-containing copolymer, wherein the amountof the polyethylene glycol is about ½ equivalent of the acid chloride.The perfluoropolyether-containing copolymer can then be separated fromthe product mixture.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a graphical representation of the dependence of the rate ofleaching of the DNA intercalating dye on the concentration of carboxylicacid IV (Krytox® 157FS_(H) (carboxylic acid XI) is used as arepresentative example);

FIG. 2 is a graphical representation of the normalized dye response withrespect to time in carboxylic acid XI, fluorosurfactant XII andHFE-7500, in accordance with an embodiment of the present disclosure;

FIG. 3 illustrates the ion-pair mediated mechanism for dye leaching,with SYBR® Green as a representative DNA intercalating dye, inaccordance with an embodiment of the present disclosure;

FIG. 4 is a graphical representation of coarse titration of a surfactantI working concentration range, in accordance with an embodiment of thepresent disclosure;

FIG. 5 is a graphical representation of the effect of Pluronic® type andconcentration on droplet stability, in accordance with an embodiment ofthe present disclosure;

FIG. 6 is a graphical representation of the effect of Pluronic® F-98concentration on droplet size, in accordance with an embodiment of thepresent disclosure;

FIG. 7 schematically illustrates a droplet generator, in accordance withan embodiment of the present disclosure;

FIG. 8A illustrates a graphical representation of a single well in theoptimized droplet digital polymerase chain reaction (ddPCR) systemcomprising 2.5 mM fluorosurfactant XII in HFE-7500 as continuous phaseand 0.50-0.75% w/w Pluronic® F-98, buffered, pH—8.3-8.4 as aqueous phaseusing EvaGreen® intercalating dye;

FIG. 8B illustrates a graphical representation for 96 wells in theoptimized ddPCR system comprising 2.5 mM fluorosurfactant XII inHFE-7500 as a continuous phase and 0.50-0.75% w/w Pluronic® F-98,buffered, pH—8.3-8.4 as aqueous phase using EvaGreen® intercalating dye,in accordance with an embodiment of the present disclosure;

FIG. 9 shows the ¹⁹F NMR spectrum of compound XI;

FIG. 10 shows the ¹³C NMR spectrum of compound XI;

FIG. 11 shows the FTIR spectrum of compound XI;

FIG. 12 shows the peaks of interest for the calculation of the averagemolecular weight of compound XI based on its ¹⁹F NMR spectrum;

FIG. 13 shows the ¹⁹F NMR spectrum of compound XII;

FIG. 14 shows the ¹³C NMR spectrum of compound XII;

FIG. 15 shows the FTIR spectrum of compound XII;

FIG. 16A shows the pseudo-first order kinetic leaching profiles for thecompound XI standards;

FIG. 16B shows the calibration curve used for calculating the purity ofcompound XII; and

FIG. 17 illustrates a graphical representation for 96 wells in theoptimized ddPCR system comprising various fluorosurfactant mixturesusing EvaGreen® intercalating dye, in accordance with an embodiment ofthe present disclosure.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed in practicing the invention.

The present disclosure provides devices, systems, methods andcompositions for using nucleic acid intercalating dyes in polymerasechain reaction assays, such as, for example, droplet digital polymerasechain reaction (ddPCR) assays. Devices, systems, methods andcompositions of the disclosure can be employed for use with varioustypes of nucleic acids, such as deoxyribonucleic acid (DNA), ribonucleicacid (RNA), and variants thereof. The devices, systems, methods andcompositions of the disclosure may prevent, reduce or inhibit theleaching of the intercalating dyes from the aqueous droplets into thecontinuous phase during PCR assays (also “dye leaching” herein). Ingeneral, the present disclosure described herein pertains to a dualphase surfactant system which enables the application of fluorescentintercalating dyes, in addition to Taqman probes, for use in PCR assays.

In some embodiments, the dual phase system is an oil phase comprising afluorosurfactant and an aqueous phase comprising a non-ionicnon-fluorosurfactant. In some cases the oil is a fluorous oil, thefluorosurfactant is a triblock polymer of Formula I, and the aqueousphase is a buffered solution of the triblock polymer PEG-PPG-PEG ofFormula VI. In some cases, the fluorosurfactant can be non-ionic. Insome cases, the fluorosurfactant can comprise a mixture of Formula I,Formula II, and/or Formula III. For example, the fluorosurfactant cancomprise a mixture of Formula I and Formula II. In certain examples, themixture can further comprise Formula XI.

In an aspect, the present disclosure provides synthetic methods toobtain high purity fluorosurfactants. These high purityfluorosurfactants may allow sufficient quantities of intercalating dyeto be maintained within droplets during droplet generation, thermalcycling, and droplet interrogation workflow. The high purityfluorosurfactants obtained herein may also facilitate a better orotherwise improved understanding of the mechanism of dye leachingphenomena.

The unexpected realization of the mechanism behind dye leaching, coupledwith the methods for production of high-purity fluorosurfactants,provide a systematic solution to a critical shortcoming of ddPCR assays.The micro-emulsion comprising the specific combination of continuousphase and aqueous phase described herein provide non-ionic stabilizeddroplets for the application of DNA intercalating dyes in ddPCR assays.

Methods of Making Fluorosurfactants

In an aspect, the present disclosure provides methods of synthesizing aperfluoropolyether-containing fluorosurfactant. Scheme 1 shows arepresentative fluorosurfactant XII of the present disclosure and anexemplary carboxylic acid XI starting material.

A method for forming a perfluoropolyether-containing fluorosurfactantcomprises (a) heating a solution of perfluoropolyether carboxylic acidin a fluorous oil in the presence of oxalyl chloride to form an acidchloride, such as perfluoropolyether acid chloride; (b) reacting theacid chloride with a polyethylene glycol to form a product mixturecomprising perfluoropolyether-containing fluorosurfactant; and (c)separating the perfluoropolyether-containing fluorosurfactant from theproduct mixture to provide a fluorosurfactant product. These syntheticoperations are summarized in Scheme 2 (see below) for thefluorosurfactant X as a representative example.

Operation 1

The conversion of perfluoropolyether carboxylic acid starting materialto perfluoropolyether acid chloride can be achieved using a variety ofreagents. In some cases this conversion is achieved by treatingperfluoropolyether carboxylic acid with oxalyl chloride (C₂O₂Cl₂). Inother cases, the conversion can be achieved by reaction ofperfluoropolyether carboxylic acid with thionyl chloride (SOCl₂). In yetother cases, phosphorous pentachloride (PCl₅) or phosphorous trichloride (PCl₃) are used to accomplish this transformation. In somecases, the conversion of the carboxylic acid starting material to thecorresponding acid chloride is achieved by reaction with carbontetrachloride and triphenyl phosphine (PPh₃). In some cases thetransformation is achieved by reaction with cyanuric chloride (C₃N₃Cl₃).

In some cases the amount of reagent used for the conversion of thecarboxylic acid starting material to the acid chloride product is in therange of about 0.1 equivalents (equiv) to 100 equiv, or about 1.0 equivto 10.0 equiv. In some cases the amount of reagent used is in the rangeof about 1.0 equiv-2.0 equiv, about 1.0 equiv-3.0 equiv, about 1.0equiv-4.0 equiv, about 1.0 equiv-5.0 equiv, about 1.0 equiv-6.0 equiv,about 1.0 equiv-7.0 equiv, about 1.0 equiv-8.0 equiv, about 1.0equiv-9.0 equiv, about 2.0 equiv-3.0 equiv, about 2.0 equiv-4.0 equiv,about 2.0 equiv-5.0 equiv, about 2.0 equiv-6.0 equiv, about 2.0equiv-7.0 equiv, about 2.0 equiv-8.0 equiv, about 2.0 equiv-9.0 equiv,about 2.0 equiv-10.0 equiv, about 3.0 equiv-4.0 equiv, about 3.0equiv-5.0 equiv, about 3.0 equiv-6.0 equiv, about 3.0 equiv-7.0 equiv,about 3.0 equiv-8.0 equiv, about 3.0 equiv-9.0 equiv, about 3.0equiv-10.0 equiv, about 4.0 equiv-5.0 equiv, about 4.0 equiv-6.0 equiv,about 4.0 equiv-7.0 equiv, about 4.0 equiv-8.0 equiv, about 4.0equiv-9.0 equiv, about 4.0 equiv-10.0 equiv, about 5.0 equiv-6.0 equiv,about 5.0 equiv-7.0 equiv, about 5.0 equiv-8.0 equiv, about 5.0equiv-9.0 equiv, about 5.0 equiv-10.0 equiv, about 6.0 equiv7.0 equiv,about 6.0 equiv-8.0 equiv, about 6.0 equiv-9.0 equiv, about 6.0equiv-10.0 equiv, about 7.0 equiv-8.0 equiv, about 7.0 equiv-9.0 equiv,about 7.0 equiv-10.0 equiv, about 8.0 equiv-9.0 equiv, about 8.0equiv-10.0 equiv, or about 9.0 equiv-10.0 equiv. In some cases theamount of reagent used is at least about 1.0, about 1.5 equiv, about 2.0equiv, about 2.5 equiv, about 3.0 equiv, about 3.5 equiv, about 4.0equiv, about 4.5 equiv, about 5.0 equiv, about 5.5 equiv, about 6.0equiv, about 6.5 equiv, about 7.0 equiv, about 7.5 equiv, about 8.0equiv, about 8.5 equiv, about 9.0 equiv, about 9.5 equiv, or about 10.0equiv. In some cases the conversion of the carboxylic acid to the acidchloride is accomplished by treatment with about 5.0 equiv of oxalylchloride.

In some cases, conversion of carboxylic acid to the corresponding acidchloride is carried out in a fluorous solvent. In some cases the solventis the perfluoroalkyl ether. In some cases the perfluoroalkyl ether ismethyl nonafluorobutyl ether (HFE-7100),1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)pentane(HFE-7300), or3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl-hexane(HFE-7500).

In some cases, conversion of the carboxylic acid to the acid chloride isfurther facilitated by adding a reaction catalyst. A catalyst is asubstance, in some cases used in small amounts relative to thereactants, that increases or otherwise modifies the rate of a reactionwithout being appreciably consumed in the process. In some cases theformation of the acid chloride is achieved by reaction with thionylchloride or oxalyl chloride, and dimethylformamide (DMF) is added as areaction catalyst. In some cases the amount of catalyst used is in therange of about 0.001 equiv to 10 equiv, or about 0.01 equiv to 1.0equiv. In some cases the amount of catalyst used is in the range ofabout 0.01 equiv-0.10 equiv, about 0.01 equiv-0.20 equiv, about 0.01equiv-0.30 equiv, about 0.01 equiv-0.40 equiv, about 0.01 equiv-0.50equiv, about 0.01 equiv-0.60 equiv, about 0.01 equiv-0.70 equiv, about0.01 equiv-0.80 equiv, about 0.01 equiv-0.90 equiv, about 0.1 equiv-0.20equiv, about 0.1 equiv-0.30 equiv, about 0.1 equiv-0.4 equiv, about 0.1equiv-0.5 equiv, about 0.1 equiv-0.6 equiv, about 0.1 equiv-0.7 equiv,about 0.1 equiv0.8 equiv, about 0.1 equiv-0.9 equiv, about 0.1 equiv-1.0equiv, about 0.2 equiv-0.3 equiv, about 0.2 equiv-0.4 equiv, about 0.2equiv-0.5 equiv, about 0.2 equiv-0.6 equiv, about 0.2 equiv-0.7 equiv,about 0.2 equiv-0.8 equiv, about 0.2 equiv-0.9 equiv, about 0.2equiv-1.0 equiv, about 0.3 equiv-0.4 equiv, about 0.3 equiv-0.5 equiv,about 0.3 equiv-0.6 equiv, about 0.3 equiv-0.7 equiv, about 0.3equiv-0.8 equiv, about 0.3 equiv-0.9 equiv, about 0.3 equiv-1.0 equiv,about 0.4 equiv-0.5 equiv, about 0.4 equiv0.6 equiv, about 0.4 equiv-0.7equiv, about 0.4 equiv-0.8 equiv, about 0.4 equiv-0.9 equiv, about 0.4equiv-1.0 equiv, about 0.5 equiv-0.6 equiv, about 0.5 equiv-0.7 equiv,about 0.5 equiv-0.8 equiv, about 0.5 equiv-0.9 equiv, about 0.5equiv-1.0 equiv, about 0.6 equiv-0.7 equiv, about 0.6 equiv-0.8 equiv,about 0.6 equiv-0.9 equiv, about 0.6 equiv-1.0 equiv, about 0.7equiv-0.8 equiv, about 0.7 equiv-0.9 equiv, about 0.7 equiv-1.0 equiv,about 0.8 equiv-0.9 equiv, about 0.8 equiv-1.0 equiv, or about 0.9equiv-1.0 equiv. In some cases the amount of the catalyst used is in therange of about 0.01 equiv-0.02 equiv, about 0.01 equiv-0.03 equiv, about0.01 equiv-0.04 equiv, about 0.01 equiv-0.05 equiv, about 0.01equiv-0.06 equiv, about 0.01 equiv-0.07 equiv, about 0.01 equiv-0.08equiv, or about 0.01 equiv-0.09 equiv. In some cases the conversion ofthe carboxylic acid to the acid chloride is accomplished by treatmentwith about 5.0 equiv of oxalyl chloride in HFE-7100 and is facilitatedby addition of about 0.05 equiv of DMF.

In some cases, formation of the acid chloride is accomplished at roomtemperature. As an alternative, the reaction is conducted at an elevatedtemperature. In some cases the reaction is carried out by heating orrefluxing the reaction mixture at the temperature in the range of about40° C.−80° C. In some cases the reaction is performed at a temperaturein the range of about 40° C.−45° C., about 40° C.−50° C., about 40°C.−55° C., about 40° C.−60° C., about 40° C.−65° C., about 40° C.−70°C., about 40° C.−75° C., about 40° C.−80° C., about 50° C.−55° C., about50° C.−60° C., about 50° C.−65° C., about 50° C.-70° C., about 50°C.−75° C., about 50° C.−80° C., about 60° C.−65° C., about 60° C.−70°C., about 60° C.−75° C., about 60° C.−80° C., about 70° C.−75° C., about70° C.−80° C., or about 75° C.-80° C. In some cases the reactiontemperature is about 40° C., about 45° C., about 50° C., about 55° C.,about 60° C., about 65° C., about 70° C., about 75° C., or about 80° C.

The reaction time for conversion of the carboxylic acid startingmaterial to the corresponding acid chloride intermediate is in the rangeof about 0.5 hours (h)-12 h. In some cases the reaction is allowed torun for a period of about 1 h-12 h, about 2 h-12 h, about 3 h-12 h,about 4 h-12 h, about 5 h-12 h, about 6 h-12 h, about 7 h-12 h, about 8h-12 h, about 9 h-12 h, about 0.5 h-11 h, about 1 h-11 h, about 2 h-11h, about 3 h-11 h, about 4 h-11 h, about 5 h-11 h, about 6 h-11 h, about7 h-11 h, about 8 h-11 h, about 9 h-11 h, about 10 h-11 h, about 0.5h-10 h, 1 h-10 h, about 2 h-10 h, about 3 h-10 h, about 4 h-10 h, about5 h-10 h, about 6 h-10 h, about 7 h-10 h, about 8 h-10 h, about 9 h-10h, about 0.5 h-9 h, about 1 h-9 h, about 2 h-9 h, about 3 h-9 h, about 4h-9 h, about 5 h-9 h, about 6 h-9 h, about 7 h-9 h, about 8 h-9 h, about0.5 h-8 h, about 1 h-8 h, about 2 h-8 h, about 3 h-8 h, about 4 h-8 h,about 5 h-8 h, about 6 h-8 h, about 7 h-8 h, 0.5 h-7 h, about 1 h-7 h,about 2 h-7 h, about 3 h-7 h, about 4 h-7 h, about 5 h-7 h, about 6 h-7h, about 0.5 h-6 h, about 1 h-6 h, about 2 h-6 h, about 3 h-6 h, about 4h-6 h, about 5 h-6 h, about 0.5 h-5 h, about 1 h-5 h, about 2 h-5 h,about 3 h-5 h, about 4 h-5 h, about 0.5 h-4 h, about 1 h-4 h, about 2h-4 h, about 3 h-4 h, about 0.5 h-3 h, about 1 h-3 h, about 2 h-3 h,about 0.5 h-2 h, about 1 h-2 h, or about 0.5 h-1 h. In some cases thereaction is allowed to run for a period of about 1 h, about 2 h, about 3h, about 4 h, about 5 h, about 6 h, about 7 h, about 8 h, about 9 h,about 10 h, about 11 h or about 12 h.

In some examples, the conversion of the carboxylic acid to the acidchloride is achieved by reaction with about 5.0 equiv of oxalyl chlorideand about 0.05 equiv of DMF as catalyst in HFE-7100 for about 4 h atabout 60° C.

In some cases the formation of the acid chloride is performed under aninert atmosphere to ensure that the acid chloride product V does notrevert back to the carboxylic acid starting material IV. In some casesthe inert atmosphere is obtained by purging the reaction flask with aninert gas. In some cases the inert gas is nitrogen or argon.

In some cases the conversion of the carboxylic acid to the acid chlorideis achieved by reaction with about 5.0 equiv of oxalyl chloride andabout 0.05 equiv of DMF as catalyst in HFE-7100 for about 4 h at about60° C. under nitrogen atmosphere.

The acid chloride obtained from this reaction can be used in the nextoperation immediately or shortly after it is prepared to prevent anydegradation or hydrolysis back to the carboxylic acid starting material.In some cases the acid chloride is stored for a time period of about 5minutes (min)-12 hour (“h”) before being used in the next operation. Insome cases the acid chloride is stored for a period of about 30 min-12h, about 1 h-12 h, about 1 h-12 h, about 2 h-12 h, about 3 h-12 h, about4 h-12 h, about 5 h-12 h, about 6 h-12 h, about 7 h-12 h, about 8 h-12h, about 9 h-12 h, about 10 h-12 h, about 11 h-12 h, about 5 min-11 h,30 min-11 h, about 1 h-11 h, about 1 h-11 h, about 2 h-11 h, about 3h-11 h, about 4 h-11 h, about 5 h-11 h, about 6 h-11 h, about 7 h-11 h,about 8 h-11 h, about 9 h-11 h, about 10 h-11 h, about 5 min-10 h, 30min-10 h, about 1 h-10 h, about 2 h-10 h, about 3 h-10 h, about 4 h-10h, about 5 h-10 h, about 6 h-10 h, about 7 h-10 h, about 8 h-10 h, about9 h-10 h, about 5 min-9 h, 30 min-9 h, about 1 h-9 h, about 2 h-9 h,about 3 h-9 h, about 4 h-9 h, about 5 h-9 h, about 6 h-9 h, about 7 h-9h, about 8 h-9 h, 5 min-8 h, 30 min-8 h, about 1 h-8 h, about 2 h-8 h,about 3 h-8 h, about 4 h-8 h, about 5 h-8 h, about 6 h-8 h, about 7 h-8h, 5 min-7 h, 30 min-7 h, about 1 h-7 h, about 2 h-7 h, about 3 h-7 h,about 4 h-7 h, about 5 h-7 h, about 6 h-7 h, about 5 min-6 h, 30 min-6h, about 1 h-6 h, about 2 h-6 h, about 3 h-6 h, about 4 h-6 h, about 5h-6 h, about 5 min-5 h, 30 min-5 h, about 1 h-5 h, about 2 h-5 h, about3 h-5 h, about 4 h-5 h, about 5 min-4 h, 30 min-4 h, about 1 h-4 h,about 2 h-4 h, about 3 h-4 h, about 5 min-3 h, 30 min-3 h, about 1 h-3h, about 2 h-3 h, about 5 min-2 h, 30 min-2 h, about 1 h-2 h, about 5min-1 h, or about 30 min-1 h before being used in the next operation. Insome cases the acid chloride is stored for a period of about 5 min,about 10 min, about 15 min, about 20 min, about 25 min, about 30 min,about 35 min, about 40 min, about 45 min, about 50 min, about 55 min,about 1.0 h, about 1.5 h, about 2.0 h, about 2.5 h, about 3.0 h, about3.5 h, about 4.0 h, about 4.5 h, about 5.0 h, about 5.5 h, about 6.0 h,about 6.5 h, about 7.0 h, about 7.5 h, about 8.0 h, about 8.5 h, about9.0 h, about 9.5 h, about 10.0 h, about 10.5 h, about 11.0 h, about 11.5h, or about 12 h prior to use in the next operation.

Operation 2

Upon formation of perfluoropolyether acid chloride, theperfluoropolyether acid chloride can be coupled to polyethylene glycol(PEG) to form a perfluoropolyether-containing fluorosurfactant. Thecoupling of the acid chloride with PEG can be achieved by reaction ofthe acid chloride with PEG, in some cases in the presence of an organicbase. In some examples, at least about 0.1 equiv, about 0.2 equiv, about0.3 equiv, about 0.4 equiv, about 0.5 equiv, about 0.6 equiv, about 0.7equiv, about 0.8 equiv, about 0.9 equiv, about 1 equiv, about 2 equiv,about 3 equiv, about 4 equiv, about 5 equiv, or about 10 equiv of PEG isused. In some cases the organic base is a tertiary amine. In an example,the amine is triethylamine. In another example, the amine isdiisopropylethylamine. In some cases the amount of amine used is in therange of 1.0 equiv-5.0 equiv. In some cases the amount of amine used isin the range of about 1.0 equiv-2.0 equiv, about 1.0 equiv-2.5 equiv,about 1.0 equiv-3.0 equiv, about 1.0 equiv-3.5 equiv, about 1.0equiv-4.0 equiv, about 1.0 equiv-4.5 equiv, about 1.0 equiv-5.0 equiv,about 1.5 equiv-2.0 equiv, about 1.5 equiv-2.5 equiv, about 1.5equiv-3.0 equiv, about 1.5 equiv-3.5 equiv, about 1.5 equiv-4.0 equiv,about 1.5 equiv-4.5 equiv, about 1.5 equiv-5.0 equiv, about 2.0 equiv2.5equiv, about 2.0 equiv-3.0 equiv, about 2.0 equiv-3.5 equiv, about 2.0equiv-4.0 equiv, about 2.0 equiv-4.5 equiv, about 2.0 equiv-5.0 equiv,about 2.5 equiv-3.0 equiv, about 2.5 equiv-3.5 equiv, about 2.5equiv-4.0 equiv, about 2.5 equiv-4.5 equiv, about 2.5 equiv-5.0 equiv,about 3.0 equiv-4.0 equiv, about 3.0 equiv-4.5 equiv, about 3.0equiv-5.0 equiv, about 3.5 equiv-4.0 equiv, about 3.5 equiv-4.5 equiv,about 3.5 equiv-5.0 equiv, about 4.0 equiv-4.5 equiv, about 4.0equiv-4.5 equiv, about 4.0 equiv4.0 equiv, or about 4.5 equiv-5.0 equiv.In some cases the amount of amine used that is used is at least about1.0 equiv, about 1.5 equiv, about 2.0 equiv, about 2.5 equiv, about 3.0equiv, about 3.5 equiv, about 4.0 equiv, about 4.5 equiv or about 5.0equiv. In some examples, about 2.0 equiv of triethylamine is used.

In some cases, coupling of the perfluoropolyether acid chloride with PEGis carried out in a fluorous solvent. In some cases the solvent is theperfluoroalkyl ether. In some cases the perfluoroalkyl ether is methylnonafluorobutyl ether (HFE-7100),1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)pentane(HFE-7300), or3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl-hexane(HFE-7500). In some cases the fluorous solvent is mixed with a secondnon-fluorous solvent. In some cases the second non-fluorous solvent isorganic ether solvent. In some cases the second solvent is diethylether, tetrahydrofuran (THF), dichloromethane (DCM), methyltetrahydrofuran and dioxane.

In some cases the ratio of the fluorous solvent to the secondnon-fluorous solvent is in the range of about 10:1 to about 1:10. Insome cases the ratio of the fluorous solvent to the second non-fluoroussolvent is about 10:1 to about 1:1. In some cases the ratio of thefluorous solvent to the second non-fluorous solvent is at least about10:1, at least about 9:1, at least about 8:1, at least about 7:1, atleast about 6:1, at least about 5:1, at least about 4:1, at least about3:1, at least about 2:1, or at least about 1:1. In some cases the ratioof the fluorous solvent to the second non-fluorous solvent is about 1:1to about 1:10. In some cases the ratio of the fluorous solvent to thesecond non-fluorous solvent is at least about 1:2, at least about 1:3,at least about 1:4, at least about 1:5, at least about 1:6, at leastabout 1:7, at least about 1:8, at least about 1:9, or at least about1:10. In some examples, the coupling of the acid chloride intermediatewith 0.5 equiv PEG is carried out in a 2:1 mixture of HFE-7100 and THF.

In some cases coupling of the perfluoropolyether acid chloride and PEGis accomplished at room temperature. As an alternative, the couplingreaction may be conducted at an elevated temperature. In some cases thereaction is carried out by heating or refluxing the reaction mixture atthe temperature in the range of about 40° C.-80° C. In some cases thereaction is performed at a temperature in the range of about 40° C.-45°C., about 40° C.-50° C., about 40° C.-55° C., about 40° C.-60° C., about40° C.-65° C., about 40° C.-70° C., about 40° C.-75° C., about 40°C.-80° C., about 50° C.−55° C., about 50° C.−60° C., about 50° C.−65°C., about 50° C.−70° C., about 50° C.−75° C., about 50° C.−80° C., about60° C.−65° C., about 60° C.−70° C., about 60° C.−75° C., about 60°C.−80° C., about 70° C.-75° C., about 70° C.−80° C., or about 75° C.−80°C. In some cases the reaction temperature is about 40° C., about 45° C.,about 50° C., about 55° C., about 60° C., about 65° C., about 70° C.,about 75° C. or about 80° C.

The reaction time for the coupling of the acid chloride and PEG can befrom about 0.1 h to 100 h, or about 1 h to 50 h. In some cases thereaction is allowed to run for about 5 h-50 h, 10 h-50 h, 15 h50 h, 20h-50 h, 25 h-50 h, 30 h-50 h, 35 h-50 h, 40 h-50 h, 45 h-50 h, 5 h-40 h,10 h-40 h, 15 h-40 h, 20 h-40 h, 25 h-40 h, 30 h-40 h, 35 h-40 h, 40h-40 h, 5 h-30 h, 10 h-30 h, 15 h-30 h, 20 h-30 h, 25 h-30 h, 5 h-20 h,10 h-20 h, 15 h-20 h, or 5 h-10 h.

In some cases the coupling of the acid chloride intermediate with halfequiv of PEG is accomplished in about 2:1 mixture of HFE-7100 and THFwith about 2.0 equiv of triethylamine at about 60° C. for about 18 h.

Upon formation of a product mixture (or crude product) comprising aperfluoropolyether-containing fluorosurfactant, theperfluoropolyether-containing fluorosurfactant can be separated from thecrude product to yield a higher purity perfluoropolyether-containingfluorosurfactant product. In some cases the purification of thefluorosurfactant product may be performed by transferring the crudeproduct into a separating funnel and dissolving it into a fluoroussolvent. The fluorous solvent may be HFE-7100, HFE-7300 or HFE-7500. Theunreacted PEG and the HCl salt of the amine (for, e.g., NEt₃.HCl, iftriethylamine is used as a base) may precipitate out during thedissolution of the crude product in fluorous solvent and float to thetop of the separation funnel. These are filtered out and the fluoroussolvent is subsequently removed to obtain the high purityfluorosurfactants.

In some cases, the solution of the crude product in fluorous solvent maybe allowed to stand for a period of about 30 min-24 h to allow all theunreacted PEG and HCl salt of the amine to precipitate out. In somecases the solution of the crude product in a fluorous solvent is allowedto stand for a time period of about 5 min-12 h before being filtered. Insome cases the solution of the crude product in a fluorous solvent isallowed to stand for a period of about 30 min-12 h, about 1 h-12 h,about 1 h-12 h, about 2 h-12 h, about 3 h-12 h, about 4 h-12 h, about 5h-12 h, about 6 h-12 h, about 7 h-12 h, about 8 h-12 h, about 9 h-12 h,about 10 h-12 h, about 11 h-12 h, about 5 min-11 h, 30 min-11 h, about 1h-11 h, about 1 h-11 h, about 2 h-11 h, about 3 h-11 h, about 4 h-11 h,about 5 h-11 h, about 6 h-11 h, about 7 h-11 h, about 8 h-11 h, about 9h-11 h, about 10 h-11 h, about 5 min-10 h, 30 min-10 h, about 1 h-10 h,about 2 h-10 h, about 3 h-10 h, about 4 h-10 h, about 5 h-10 h, about 6h-10 h, about 7 h-10 h, about 8 h-10 h, about 9 h-10 h, about 5 min-9 h,30 min-9 h, about 1 h-9 h, about 2 h-9 h, about 3 h-9 h, about 4 h-9 h,about 5 h-9 h, about 6 h-9 h, about 7 h-9 h, about 8 h-9 h, 5 min h-8 h,30 min h-8 h, about 1 h-8 h, about 2 h-8 h, about 3 h-8 h, about 4 h-8h, about 5 h-8 h, about 6 h-8 h, about 7 h-8 h, 5 min-7 h, 30 min-7 h,about 1 h-7 h, about 2 h-7 h, about 3 h-7 h, about 4 h-7 h, about 5 h-7h, about 6 h-7 h, about 5 min-6 h, 30 min-6 h, about 1 h-6 h, about 2h-6 h, about 3 h-6 h, about 4 h-6 h, about 5 h-6 h, about 5 min-5 h, 30min-5 h, about 1 h-5 h, about 2 h-5 h, about 3 h-5 h, about 4 h-5 h,about 5 min-4 h, 30 min-4 h, about 1 h-4 h, about 2 h-4 h, about 3 h-4h, about 5 min-3 h, 30 min-3 h, about 1 h-3 h, about 2 h-3 h, about 5min-2 h, 30 min-2 h, about 1 h-2 h, about 5 min-1 h, or about 30 min-1 hbefore being used in the next operation (e.g., separation). In somecases the solution of the crude product in a fluorous solvent is allowedto stand for a period of about 5 min, about 10 min, about 15 min, about20 min, about 25 min, about 30 min, about 35 min, about 40 min, about 45min, about 50 min, about 55 min, about 1.0 h, about 1.5 h, about 2.0 h,about 2.5 h, about 3.0 h, about 3.5 h, about 4.0 h, about 4.5 h, about5.0 h, about 5.5 h, about 6.0 h, about 6.5 h, about 7.0 h, about 7.5 h,about 8.0 h, about 8.5 h, about 9.0 h, about 9.5 h, about 10.0 h, about10.5 h, about 11.0 h, about 11.5 h, or about 12 h prior to beingfiltered. In some further cases the crude product in a fluorous solventis allowed to stand for a period of about 13 h, about 14 h, about 15 h,about 16 h, about 17 h, about 18 h, about 19 h, about 20 h, about 21 h,about 22 h, about 23 h, or about 24 h.

The purity of the fluorosurfactant can be assayed by numerous analyticaltechniques including but not limited to ¹⁹F NMR, ¹H NMR, ¹³C NMR, FTIR,elemental analysis and a variety of fluorescence techniques.

Of particular interest is the resonance due to the carbonyl carbon (C=0)in the ¹³C spectrum of product I (˜157 ppm), this resonance hasundergone a significant up-field shift when compared to the carbonylcarbon of the carboxylic acid present in the carboxylic acid startingmaterial (˜162 ppm). In the ¹³C NMR spectrum of the purified product, arelatively weak resonance present at about present at 157±2 ppm may beassigned to the carbonyl carbons (C=0) of the two ester linkages, whilethe resonance due to the carbonyl carbon (COOH) of the carboxylic acidstarting material IV observed at 162±2 ppm must be absent from thespectrum.

Dye Leaching

The use of common DNA intercalating dyes in ddPCR systems is currentlydifficult due to the leaching of the intercalating dyes from within thedroplet (aqueous phase) into the continuous (or oil) phase. In somesituations, a decreased response in droplet fluorescence is observedover time, while the background fluorescence increases. A system thatenables the use of DNA intercalating dyes in ddPCR reactions, whileminimizing the dye leaching, may advantageously enable convenient andeconomical use of ddPCR assays. Thus, recognized herein is a need of adual phase system that enables the application of fluorescent DNA dyesfor use in ddPCR assays.

The present disclosure provides a mechanism for the dye-leachingphenomenon observed during the use of DNA intercalating dyes in ddPCRassays. This migration of the intercalating dyes from the aqueous phasedroplets to the continuous phase has generally been described as micellemediated transport or as passive diffusion. Applicants have recognizedthat dye leaching may be unexpectedly due to the presence of residualcarboxylic acid material during the synthesis of surfactant. Withoutbeing bound by theory, a majority of the fluorosurfactants used in ddPCRassays are synthesized from carboxylic acid terminated startingmaterials. Inefficient conversion of the carboxylic acid startingmaterial to the corresponding ester, amide or alternate carbonylcompound may result in unconverted carboxylic acid as being present asimpurity in the fluorosurfactant. As depicted in FIG. 1, the amount ofdye leaching is found to increase with the amount of the unreactedcarboxylic acid impurity in the fluorosurfactant.

With respect to some of the high purity fluorosurfactant compositions ofthe present disclosure, carboxylic acid impurity is reduced or absentand the dye leaching is minimized (FIG. 2). These results may point toan alternate mechanism of dye-leaching, such that the amount of dyeleaching is dependent upon the amount of the carboxylic acid impuritypresent in the fluorosurfactant. The new mechanism for dye leaching isschematically illustrated in FIG. 3.

In some cases, in the beginning of the ddPCR reaction, the carboxylicacid impurity is present in the continuous phase or the oil phase while,the DNA intercalating dye is present in the aqueous phase. Once thedroplets are generated, the carboxylic acid compound enters the aqueousphase, where it is deprotonated. The resulting carboxylate ion thenion-pairs with the cationic intercalating dye and the neutralcarboxylate/dye complex then migrates across the phase boundary into thefluorous phase. This results in a decreased relative response inobserved droplet fluorescence over time, while the backgroundfluorescence increases over time. The ion-pairing approach may be an‘active’ transport mechanism, which, in some cases, may be favored overany micelle mediated transport. The ion-pair mechanism may also explainthe high rate of dye leaching where significant quantity of dye is lostfrom the aqueous phase in just minutes, for example.

Fluorosurfactant

A surfactant may be characterized as a surface-active agent capable ofreducing the surface tension of a liquid in which it is dissolved,and/or the interfacial tension with another phase. A surfactant mayincorporate both a hydrophilic portion and a hydrophobic portion, whichmay collectively confer a dual hydrophilic-hydrophobic character on thesurfactant. Fluorosurfactants, or fluorinated surfactants, areorganofluorine chemical compounds that have multiple fluorine atoms.They can be either polyfluorinated or perfluorinated. In some cases, thefluorosurfactant can be non-ionic.

An aspect of the present disclosure provides a fluorous surfactant ofFormula I. The surfactant of Formula I is a triblock copolymercomprising polyethylene glycol (PEG) polymer covalently linked topolyhexafluoropropylene oxide (PFPE) at both ends. In an example, thecovalent linkage between the PEG block and PFPE blocks at both the endsis an amide linkage (A and B are nitrogen). In another example, thelinkage between the PEG block and the two PFPE blocks is by ester bonds(A and B are oxygen). In another example, the PEG block can be linked tothe PFPE blocks by an amide bond at one end and an ester bond at theother end (A is oxygen, B is nitrogen; or A is nitrogen, B is oxygen).

The lengths of the PFPE chains and the PEG block may be important indetermining the properties of the fluorosurfactant. In some aspects ofthe present disclosure both m₁ and m₂ are independently in a range ofabout 10-100. In some cases both m₁ and m₂ are independently in therange of about 10-20, about 10-30, about 10-40, about 10-50, about10-60, about 10-70, about 10-80, about 10-90, about 20-30, about 20-40,about 20-50; about 20-60, about 20-70, about 20-80, about 20-90, about20-100, about 30-40, about 30-50, about 30-60, about 30-70, about 30-80,about 30-90, about 30-100, about 40-50, about 40-60, about 40-70, about40-80, about 40-90, about 40-100, about 50-60, about 50-70, about 50-80,about 50-90, about 50-100, about 60-70, about 60-80, about 60-90, about60-100, about 70-80, about 70-90, about 70-100, about 80-90, about80-100, or about 90-100. In some cases m₁ is in the range of about10-20, about 10-30, about 10-40, about 10-50, about 10-60, about 10-70,about 10-80, about 10-90, about 20-30, about 20-40, about 20-50; about20-60, about 20-70, about 20-80, about 20-90, about 20-100, about 30-40,about 30-50, 30-60, about 30-70, about 30-80, about 30-90, about 30-100,about 40-50, about 40-60, about 40-70, about 40-80, about 40-90, about40-100, about 50-60, about 50-70, about 50-80, about 50-90, about50-100, about 60-70, about 60-80, about 60-90, about 60-100, about70-80, about 70-90, about 70-100, about 80-90, about 80-100, or about90-100. In some cases m₂ is in the range of about 10-20, about 10-30,about 10-40, about 10-50, about 10-60, about 10-70, about 10-80, about10-90, about 20-30, about 20-40, about 20-50; about 20-60, about 20-70,about 20-80, about 20-90, about 20-100, about 30-40, about 30-50, about30-60, about 30-70, about 30-80, about 30-90, about 30-100, about 40-50,about 40-60, about 40-70, about 40-80, about 40-90, about 40100, about50-60, about 50-70, about 50-80, about 50-90, about 50-100, about 60-70,about 60-80, about 60-90, about 60-100, about 70-80, about 70-90, about70-100, about 80-90, about 80-100, or about 90-100.

The value of ‘n’ in the present disclosure may be in a range of about10-60. In some cases the value of ‘n’ is in the range of about 10-20,about 10-30, about 10-40, about 10-50, about 15-20, about 15-30, about15-40, about 15-50, about 15-60, about 20-30, about 20-40, about 20-50,about 20-60, about 25-30, about 25-40, about 25-50, about 25-60, about30-40, about 30-50, about 30-60, about 3540, about 35-50, about 35-60,about 40-50, about 40-60, about 45-50, about 45-60, about 50-50, about55-60. In some embodiments, each of m₁ and m₂ is about 35 and ‘n’ isabout 22.

Another aspect of the current disclosure provides a high purityfluorosurfactant of Formula II. The surfactant II is a diblock copolymerof PEG and PFPE. The value of m₃, which represents the length of thePFPE block, is in a range of about 10-100. In some aspects, m₃ is in therange of about 1020, about 10-30, about 10-40, about 10-50, about 10-60,about 10-70, about 10-80, about 10-90, about 20-30, about 20-40, about20-50; about 20-60, about 20-70, about 20-80, about 20-90, about 20-100,about 30-40, about 30-50, about 30-60, about 30-70, about 30-80, about30-90, about 30-100, about 40-50, about 40-60, about 40-70, about 40-80,about 40-90, about 40-100, about 50-60, about 50-70, about 50-80, about50-90, about 50-100, about 60-70, about 60-80, about 60-90, about60-100, about 70-80, about 70-90, about 70-100, about 80-90, about80-100, or about 90-100. The length of the PEG unit, ‘n’ is in the rangeof about 10-60. In some cases the value of ‘n’ is in the range of about10-20, about 10-30, about 10-40, about 10-50, about 15-20, about 15-30,about 15-40, about 15-50, about 15-60, about 20-30, about 20-40, about20-50, about 20-60, about 25-30, about 25-40, about 25-50, about 25-60,about 30-40, about 30-50, about 30-60, about 35-40, about 35-50, about35-60, about 40-50, about 40-60, about 45-50, about 45-60, about 50-50,or about 55-60. The end of the PEG block, —OR, can either be a hydroxylgroup (i.e., —OH), an alkoxy group, or an amine—that is, R can behydrogen, an alkyl group, or an amine.

Another aspect of the current disclosure provides a high purityfluorosurfactant of Formula III. The surfactant of Formula III is atriblock copolymer wherein two units PEG (same or different length) andone unit of PFPE are connected by a phosphate linker (PO₄). The lengthof the PFPE chain m₄ is in a range of about 10-100. In some cases m₄ isin the range of about 10-20, about 10-30, about 10-40, about 10-50,about 10-60, about 10-70, about 10-80, about 10-90, about 20-30, about20-40, about 20-50; about 20-60, about 20-70, about 20-80, about 20-90,about 20-100, about 30-40, about 30-50, about 30-60, about 30-70, about30-80, about 30-90, about 30-100, about 40-50, about 40-60, about 40-70,about 40-80, about 40-90, about 40-100, about 50-60, about 50-70, about50-80, about 50-90, about 50-100, about 60-70, about 60-80, about 60-90,about 60-100, about 70-80, about 70-90, about 70-100, about 80-90, about80-100, or about 90-100. The lengths of the two PEG units, n₃ and n₄ areindependently in a range of about 10-60. In some cases n₃ and n₄ areindependently in the range of about 10-20, about 10-30, about 10-40,about 10-50, about 15-20, about 15-30, about 15-40, about 15-50, about15-60, about 20-30, about 20-40, about 20-50, about 20-60, about 25-30,about 25-40, about 25-50, about 25-60, about 30-40, about 30-50, about30-60, about 35-40, about 35-50, about 35-60, about 40-50, about 40-60,about 45-50, about 45-60, about 50-50, or about 55-60. In some cases n₃is in the range of about 10-20, about 10-30, about 10-40, about 10-50,about 15-20, about 15-30, about 15-40, 15-50, about 15-60, about 20-30,about 20-40, about 20-50, about 20-60, about 25-30, about 25-40, about25-50, about 25-60, about 30-40, about 30-50, about 30-60, about 35-40,about 35-50, about 35-60, about 40-50, about 40-60, about 45-50, about45-60, about 50-50, or about 55-60. In some cases n₄ is in the range ofabout 10-20, about 10-30, about 10-40, about 10-50, about 15-20, about15-30, about 15-40, about 15-50, about 15-60, about 20-30, about 20-40,about 20-50, about 20-60, about 25-30, about 25-40, about 2550, about25-60, about 30-40, about 30-50, about 30-60, about 35-40, about 35-50,about 35-60, about 40-50, about 40-60, about 45-50, about 45-60, about50-50, or about 55-60. The CH₂ spacer (n₅) can be 0, 1, 2 or 3 carbonsin length.

In some cases, the fluorosurfactant can comprise a mixture of Formula I,Formula II, and/or Formula III. In certain cases, the fluorosurfactantcan comprise a mixture of Formula I and Formula II. In some examples,the fluorosurfactant can comprise at least about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, orabout 99.9% (w/w) of a mixture of Formula I and Formula II. For example,the fluorosurfactant can comprise at least about 80%, about 90%, or 95%(w/w) of a mixture of Formula I and Formula II.

Further, the mixture of Formula I and Formula II can be in a ratio ofgreater than about 1:199, about 1:99, about 2:98, about 3:97, about4:96, about 5:95, about 10:90, about 15:85: about 20:80, about 25:75,about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20,about 85:15, about 90:10, about 95:5, about 96:4, about 97:3, about98:2, about 99:1, or about 199:1 (w/w). For example, the mixture ofFormula I and Formula II can be in a ratio of greater than about 80:20,about 90:10, or about 95:5 (w/w).

Alternatively, the mixture of Formula I and Formula II can be in a ratioof less than about 1:199, about 1:99, about 2:98, about 3:97, about4:96, about 5:95, about 10:90, about 15:85: about 20:80, about 25:75,about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20,about 85:15, about 90:10, about 95:5, about 96:4, about 97:3, about98:2, about 99:1, or about 199:1 (w/w). For example, the mixture ofFormula I and Formula II can be in a ratio of less than about 80:20,about 90:10, or about 95:5 (w/w).

In certain cases, the fluorosurfactant mixture can further compriseFormula XI:

wherein n can be about 10 to 100, about 10 to 80, about 15 to 80, about15 to 50, or about 20 to 50. For example, the fluorosurfactant mixturecan comprise about 0.1% to 50%, about 0.1% to 20%, about 0.2% to 20%,about 0.2% to 10%, about 0.5% to 10%, about 0.5% to 5%, or about 1% to5% (w/w) of Formula XI.

Further, the fluorosurfactant mixture can comprise about greater thanabout 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%,about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45% or about 50% (w/w) ofFormula XI. For example, the fluorosurfactant mixture can comprise aboutgreater than about 0.1%, about 0.5%, about 1%, about 2%, or about 5%(w/w) of Formula XI.

Alternatively, the fluorosurfactant mixture can comprise about less thanabout 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%,about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45% or about 50% (w/w) ofFormula XI. For example, the fluorosurfactant mixture can comprise aboutless than about 1%, about 2%, about 5%, about 10%, or about 20% (w/w) ofFormula XI.

In certain cases, the oil formulation can comprise less than about 0.1%,about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%,about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 2.5%,about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45% or about 50% (w/w) of FormulaXI. For example, the oil formulation can comprise less than about 1%,about 2%, about 5%, about 10%, or about 20% (w/w) of Formula XI.

In other cases, the oil formulation can comprise greater than about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45% or about 50% (w/w) ofFormula XI. For example, the oil formulation can comprise greater thanabout 0.1%, about 0.5%, about 1%, about 2%, or about 5% (w/w) of FormulaXI.

A surfactant may be characterized according to a Hydrophile-LipophileBalance (HLB) value, which may be defined as the ratio of the molecularweight (MW) of the hydrophilic portion of the compound to the total MWof the compound. HLB may be controlled by varying the lengths/MWs of thehydrophobic portion (PFPE) and the hydrophilic portion (PEG) of themolecule. HLB of the fluorosurfactant may play a critical role in‘anchoring’ the surfactant into the aqueous droplet. For example andwithout being bound to theory, decreasing the specified PEG MW mayresult in thermal instability due to an increased rate of exchange ofsurfactant at the droplet boundary, while increasing the optimized PEGMW may result in the generation of non-uniform, poly-disperse dropletpopulations. Furthermore, decreasing the specified PFPE molecular weight(MW) may result in loss of thermal stability due to coalescence (lessdense fluorophilic brush network). In general, the longer (higher MW)the perfluoropolyether chain the greater the system's resistance tothermally induced droplet coalescence. In some embodiments, the MW ofthe perfluoropolyether chain is at least about 3000, at least about4000, at least about 5000, at least about 6000, at least about 7000, atleast about 8000, at least about 9000, or at least about 10000. In someother embodiments, the MW of the perfluoropolyether chain is about 3000,about 4000, about 5000, about 6000, about 7000, about 8000, about 9000,or about 10000.

In some aspects of the current disclosure, the HLB value of thefluorosurfactant is in the range of about 0-20. In some cases, the HLBvalues of the non-ionic surfactant ranges from about 0-10, about 0-20,about 5-10, about 5-15, about 5-20, or about 10-20. In some furtheraspects, the HLB value of the fluorosurfactant is about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, or about 20.

In general, the fluorosurfactants obtained in the present disclosure arehigh purity fluorosurfactants and can be successfully used in ddPCRassays with intercalating dyes while avoiding dye leaching. In somecases, the fluorosurfactants can be greater than about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 85.5%, about 86%, about 86.5%,about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about 89.5%,about 90%, about 90.5%, about 91.5%, about 92%, about 92.5%, about 93%,about 93.5%, about 94%, about 94.5%, about 95%, about 96.5%, about 97%,about 97.5%, about 98%, about 98.5%, about 99%, or about 99.5% weightpercent (w/w) pure. In some examples, the fluorosurfactants are greaterthan about 90% weight percent (w/w) pure. For example, thefluorosurfactants can comprise at least 90% (w/w) of a mixture ofFormula I and Formula II. In some cases the weight percent purity of thefluorosurfactant is in the range of about 90%-91% w/w, about 90%-92%w/w, about 90%-93% w/w, about 90%-94% w/w, about 90%-95% w/w, about90%-96% w/w, about 90%-97% w/w, about 90%-98% w/w, about 90%-99% w/w,about 91%92% w/w, about 91%-93% w/w, about 91%-94% w/w, about 91%-95%w/w, about 91%-96% w/w, about 91%-97% w/w, about 91%-98% w/w, 91%-99%w/w, about 92%-93% w/w, about 92%-94% w/w, about 92%-95% w/w, about92%-96% w/w, about 92%-97% w/w, about 92%-98% w/w, about 92%99% w/w,about 93%-94% w/w, about 93%-95% w/w, about 93%-96% w/w, about 93%-97%w/w, about 93%-98% w/w, about 93%-99% w/w, about 94%-95% w/w, about94%-96% w/w, about 94%-97% w/w, about 94%-98% w/w, or about 94%-99% w/w.In some cases the fluorosurfactants are greater than about 95% (weightpercent) pure. For example, the fluorosurfactants can comprise at least95% (w/w) of a mixture of Formula I and Formula II. In some cases theweight percent purity of the fluorosurfactant is in the range of about95%-96% w/w, about 95%-97% w/w, about 95%-98% w/w, about 95%-99% w/w,about 96%-97% w/w, about 96%-98% w/w, about 96%-99% w/w, about 97%-98%w/w, about 97%-99% w/w, or about 98%-99% w/w. In some cases, thefluorosurfactants may have weight percent purity of about 90% w/w, about91% w/w, about 92% w/w, about 93% w/w, about 94% w/w, about 95% w/w,about 96% w/w, about 97% w/w, about 98% w/w, or about 99% w/w.

The concentration of fluorosurfactant may be important for dropletstability. In some cases, the concentration of fluorosurfactant may bein a range of 0.1-10.0 mM (millimolar). In some embodiments, theconcentration of fluorosurfactant is in a range of about 0.1 mM-1.0 mM,about 0.1 mM-2.0 mM, about 0.1 mM-3.0 mM, about 0.1 mM-4.0 mM, about 0.1mM-5.0 mM, about 0.1 mM-6.0 mM, about 0.1 mM-7.0 mM, about 0.1 mM-8.0mM, about 0.1 mM-9.0 mM, about 0.5 mM-1.0 mM, about 0.5 mM-2.0 mM, about0.5 mM-3.0 mM, about 0.5 mM-4.0 mM, about 0.5 mM-5.0 mM, about 0.5mM-6.0 mM, about 0.5 mM-7.0 mM, about 0.5 mM-8.0 mM, about 0.5 mM-9.0mM, about 0.5 mM-10.0 mM, about 1.0 mM-2.0 mM, about 1.0 mM-3.0 mM,about 1.0 mM-4.0 mM, about 1.0 mM-5.0 mM, about 1.0 mM-6.0 mM, about 1.0mM-7.0 mM, about 1.0 mM-8.0 mM, about 1.0 mM-9.0 mM, about 1.0 mM-10.0mM, about 1.5 mM-2.0 mM, about 1.5 mM-3.0 mM, about 1.5 mM-4.0 mM, about1.5 mM5.0 mM, about 1.5 mM-6.0 mM, about 1.5 mM-7.0 mM, about 1.5 mM-8.0mM, about 1.5 mM-9.0 mM, about 1.5 mM-10.0 mM, about 2.0 mM-3.0 mM,about 2.0 mM-4.0 mM, about 2.0 mM-5.0 mM, about 2.0 mM-6.0 mM, about 2.0mM-7.0 mM, about 2.0 mM-8.0 mM, about 2.0 mM-9.0 mM, about 2.0 mM-10.0mM, about 2.5 mM-3.0 mM, about 2.5 mM-4.0 mM, about 2.5 mM-5.0 mM, about2.5 mM6.0 mM, about 2.5 mM-7.0 mM, about 2.5 mM-8.0 mM, about 2.5 mM-9.0mM, about 2.5 mM-10.0 mM, about 3.0 mM-4.0 mM, about 3.0 mM-5.0 mM,about 3.0 mM-6.0 mM, about 3.0 mM-7.0 mM, about 3.0 mM-8.0 mM, about 3.0mM-9.0 mM, about 3.0 mM-10.0 mM, about 3.5 mM-4.0 mM, about 3.5 mM-5.0mM, about 3.5 mM-6.0 mM, about 3.5 mM-7.0 mM, about 3.5 mM-8.0 mM, about3.5 mM-9.0 mM, about 3.5 mM-10.0 mM, about 4.0 mM-5.0 mM, about 4.0mM-6.0 mM, about 4.0 mM7.0 mM, about 4.0 mM-8.0 mM, about 4.0 mM-9.0 mM,about 4.0 mM-10.0 mM, about 4.5 mM-5.0 mM, about 4.5 mM-6.0 mM, about4.5 mM-7.0 mM, about 4.5 mM-8.0 mM, about 4.5 mM-9.0 mM, about 4.5mM-10.0 mM, about 5.0 mM-6.0 mM, about 5.0 mM-7.0 mM, about 5.0 mM-8.0mM, about 5.0 mM-9.0 mM, about 5.0 mM-10.0 mM, about 5.5 mM-6.0 mM,about 5.5 mM-7.0 mM, about 5.5 mM-8.0 mM, about 5.5 mM-9.0 mM, about 5.5mM-10.0 mM, about 6.0 mM-7.0 mM, about 6.0 mM8.0 mM, about 6.0 mM-9.0mM, about 6.0 mM-10.0 mM, about 6.5 mM-7.0 mM, about 6.5 mM-8.0 mM,about 6.5 mM-9.0 mM, about 6.5 mM-10.0 mM, about 7.0 mM-8.0 mM, about7.0 mM-9.0 mM, about 7.0 mM-10.0 mM, about 7.5 mM-8.0 mM, about 7.5mM-9.0 mM, about 7.5 mM-10.0 mM, about 8.0 mM-9.0 mM, about 8.0 mM-10.0mM, about 8.5 mM-9.0 mM, about 8.5 mM-10.0 mM, about 9.0 mM-10.0 mM, orabout 9.5 mM-10.0 mM. In some other embodiments, the concentration offluorosurfactant is about 0.5 mM, about 1.0 mM, about 1.5 mM, about 2.0mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5mM, about, 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, or about 9.5mM.

Oil Phase

The oil phase or the continuous phase used in this disclosure is anyliquid compound or mixture of liquid compounds that is immiscible withwater. The oil used may be or include at least one of silicone oil,mineral oil, hydrocarbon oil, fluorocarbon oil, vegetable oil, or acombination thereof, among others. Any other suitable components mayalso be present in the oil phase, such as at least one surfactant,reagent, other additive, preservative, particles, or any combinationthereof.

In some cases, the oil is fluorinated oil. Fluorinated oil can be anyfluorinated organic compound. In some cases, the fluorinated oil isperfluorocarbon, such as perfluorooctane or perfluorohexane. In somecases, the fluorine-containing compound is a partially fluorinatedhydrocarbon, such as 1,1,1-trifluorooctane or1,1,1,2,2-petantafluorodecane. The fluorinated organics can be linear,cyclic or heterocyclic. In addition to carbon and fluorine, thefluorinated organic compound can further contain hydrogen, oxygen,nitrogen, sulfur, chlorine, bromine atoms.

In some cases, the fluorinated oil is a perfluoroalkyl ether like methylnonafluoroisobutyl ether sold as NOVEC™ HFE-7100 Engineered Fluid. Insome cases, the fluorine-containing compound is3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl-hexane,sold as NOVEC™ HFE-7500 Engineered Fluid. In some cases, thefluorine-containing compound is3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl-hexane,sold as NOVEC™ HFE-7500 Engineered Fluid. In some cases, thefluorine-containing compound can be1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-trifluoromethyl-pentane, soldas NOVEC™ HFE-7300 Engineered Fluid.

In the methods of the present disclosure, the fluorosurfactants residemainly in the oil phase. In some cases at least about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, or about 99% of the fluorosurfactant resides in the oil phase.

Aqueous Phase

The aqueous phase can be any liquid miscible with water. That is,aqueous phase can be any liquid that when mixed with water at roomtemperature, forms a stable single phase solution. In some embodimentsthe aqueous phase can comprise one or more physiologically acceptablereagents and/or solvents etc. Some non-limiting examples of aqueousphase include water, DMF, DMSO, methanol or ethanol.

In some cases, the aqueous phase can be a buffer solution. The bufferedsolution can comprise of Tris and KCl. In some cases, the concentrationof Tris is about, more than about, or less than about 1 mM, 5 mM, 10 mM,15 mM, 20 mM, 30 mM, 50 mM, 80 mM, 160 mM, or 200 mM. In some cases, theconcentration of potassium chloride can be about, more than about, orless than about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50mM, about 60 mM, about 80 mM, about 100 mM, about 200 mM. The bufferedsolution can comprise about 160 mM Tris and about 40 mM KCl. In somecases, magnesium chloride (MgCl₂) is added to an amplification reactionat a concentration of about, more than about, or less than about 1.0 mM,about 2.0 mM, about 3.0 mM, about 4.0 mM, or about 5.0 mM.

In some cases the pH of the buffered aqueous phase is in the range ofabout 7.0-10.0. In some cases the pH of the aqueous phase is in therange of about 7.5-10.0, about 8.0-10.0, about 8.5-10.0, about 9.0-10.0,about 9.5-10.0, about 7.0-9.5, about 7.5-9.5, about 8.0-9.5, about8.5-9.5, about 9.0-9.5, about 7.0-9.0, about 7.5-9.0, about 8.0-9.0,about 8.5-9.0, about 7.0-8.5, about 7.5-8.5, about 8-8.5, about 7.0-8.0,or about 7.5-8.0. In some cases the pH of the aqueous solution is about7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6,about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9,about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about9.6, about 9.7, about 9.8, about 9.9, or about 10.

Non-Ionic Non-Fluorosurfactant

The aqueous phase of the present disclosure further comprises anon-ionic non-fluorosurfactant. Surfactant is a surface active substancecapable of reducing the surface tension of a liquid in which it ispresent. A surfactant, which also or alternatively may be described as adetergent and/or wetting agent, may incorporate both a hydrophilicportion and a hydrophobic portion, which may collectively confer a dualhydrophilic-hydrophobic character on the surfactant. A surfactant may,in some cases, be characterized according to its hydrophilicity relativeto its hydrophobicity. In some embodiments, the non-ionicnon-fluorosurfactant is a polyalkylene oxide block copolymer surfactant.Particularly useful are block copolymers of polypropylene glycol(H—(O—CH(CH₃)—CH₂)_(n)—OH, PPG) and polyethylene glycol(H—(O—CH₂—CH₂)_(n)—OH, PEG) having the general formula[PPG_(n)-PEG_(m)].

The non-ionic non-fluorosurfactant could be a di, tri, tetra, penta oreven higher block polymer of PEG and PPG. In some cases the non-ionicnon-fluorosurfactant is an alternating copolymer with regularalternating PEG and PPG units of general formula (PEG-PPG)_(x), whereinx is in the range of about 10-100. In some cases, x is in the range ofabout 10-20, about 10-30, about 10-40, about 10-50, about 10-60, about10-70, about 10-80, about 10-90, about 20-30, about 20-40, about 20-50,about 20-60, about 20-70, about 20-80, about 20-90, about 20-100, about30-40, about 30-50, about 30-60, about 30-70, about 30-80, about 30-90,about 30-100, about 40-50, about 40-60, about 40-70, about 40-80, about40-90, about 40-100, about 50-60, about 50-70, about 50-80, about 50-90,about 50-100, about 60-70, about 60-80, about 60-90, about 60-100, about70-80, about 70-90, about 70-100, about 80-90, about 80-100, or about90-100. In some cases the non-ionic non-fluorosurfactant is a randomcopolymer of PEG and PPG with the monomeric PEG and PPG blocks attachedin a random sequence.

In some cases the molecular weight of PEG and PPG block copolymer is inthe range of about 4,000 dalton (“Da”)-25,000 Da. In some cases themolecular weight of the non-ionic non-fluorosurfactant is in the rangeof about 10,000 Da-25000 Da, about 15,000 Da-25,000 Da, about 20,000Da-25,000 Da, about 4,000 Da-20,000 Da, about 10,000 Da-20000 Da, about15,000 Da-20,000 Da, about 4,000 Da-15,000 Da, about 10,000 Da-15,000Da, or about 4,000 Da-10,000 Da. In some cases the molecular weight ofthe non-ionic non-fluorosurfactant is about 4,000 Da, 4,500 Da, about5,000 Da, about 5,500 Da, about 6,000 Da, about 6,500 Da, about 7,000Da, about 7,500 Da, about 8,000 Da, about 8,500 Da, about 9,000 Da,about 9,500 Da, about 10,000 Da, about 10,500 Da, about 11,000 Da, about11,500 Da, about 12,000 Da, about 12,500 Da, about 13,000 Da, about13,500 Da, about 14,000 Da, about 14,500 Da, about 15,000 Da, about15,500 Da, about 16,000 Da, about 16,500 Da, about 17,000 Da, about17,500 Da, about 18,000 Da, about 18,500 Da, about 19,000 Da, about19,500 Da, about 20,000 Da, about 20,500 Da, about 21,000 Da, about21,500 Da, about 22,000 Da, about 22,500 Da, about 23,000 Da, about23,500 Da, about 24,000 Da, about 24,500 Da or about 25,000 Da.

In some cases, the non-ionic non-fluorosurfactant is a triblockcopolymer of polypropylene oxide and polyethylene oxide (Scheme 3,Formula VI, see below) sold under the trade names Pluronic® (registeredtrademark of BASF Corporation, also known under non-proprietary name“poloxamers”). These copolymers consist of a central hydrophobic chainof PPG flanked by two hydrophilic chains of PEG (PEG-PPG-PEG). The blockcopolymers with varying length of the individual PEG and PPG units arecharacterized by different hydrophilic-lipophilic balance (HLB). In someembodiments, the Pluronic® surfactant is Pluronic® F-38, Pluronic® F-68,Pluronic® F-77, Pluronic® F-87, Pluronic® F-88, Pluronic®F-98, Pluronic®F-108 or Pluronic® F-127 (a=101, b=56).

In further embodiments of the disclosure the non-ionic non-fluoroussurfactant is Nonidet® P40 (registered trade mark of Shell ChemicalCo.). The general structure of Nonidet® comprises of a hydrophilicpolyethylene chain and an aromatic hydrocarbon lypophilic group (Scheme3, Formula VII, see below). In some cases the non-ionic non-fluoroussurfactant is Nonidet® P40.

In further embodiments of the disclosure the non-ionic non-fluoroussurfactant can be other polyethylene glycol derivatives includingTriton® X100 (registered trademark of Union Carbide Corp.). The generalstructure of Triton® comprises of a hydrophilic polyethylene chain andan aromatic hydrocarbon lipophilic group (Scheme 3, Formula VIII, seebelow). In some cases the non-ionic non-fluorous surfactant is Triton®X-15 (x=1.5 avg), Triton® X-35 (x=3 avg), Triton® X-45 (x=4.5 avg),Triton® X-100 (x=9.5 avg), Triton® X-102 (x=12 avg), Triton® X-114(x=7.5 avg), Triton® X-165 (x=16 avg), Triton® X-305 (x=30 avg), Triton®X-405 (x=35 avg), or Triton® X-705 (x=1.5 avg).

In further embodiments of the disclosure the non-ionic non-fluoroussurfactant is a polyoxyethylene derivative of sorbitan monolaurate(Scheme 3, Formula IX, see below), commercially available as Tween®(registered trademark of Uniquema Americas LLC). In some cases, thenon-ionic non-fluorous surfactant is Tween® 20 (R═CH₂(CH₂)₉CH₃)), Tween®40 (R═CH₂(CH₂)₁₃CH₃), Tween® 60 (R═CH₂(CH₂)₁₅CH₃) or Tween®-80(R═(CH₂)₇CH═CH(CH₂)₈.

The concentration of non-ionic non-fluorosurfactant may be in a range ofabout 0.1-5.0% weight percent. In some embodiments the concentration ofthe non-ionic non-fluorosurfactant is less than about 1.5% weight byweight (“w/w”). In some cases, the concentration of Pluronic® is lessthan about 1.4% w/w, about 1.3% w/w, about 1.2% w/w, about 1.1% w/w,about 1.0% w/w, about 0.9% w/w, about 0.8% w/w, about 0.7% w/w, about0.6% w/w, about 0.5% w/w, about 0.4% w/w, about 0.3% w/w, about 0.2% w/wor about 0.1% w/w. In some embodiments the Pluronic® F-98 concentrationis in the range of about 0.50% w/w-1.5% w/w. In some embodiments thePluronic® F-98 concentration is in the range of about 0.50% w/w-0.60%w/w, about 0.50% w/w-0.65% w/w, about 0.50% w/w-0.70% w/w, about 0.55%w/w-0.60% w/w, about 0.55% w/w-0.65% w/w, about 0.55% w/w-0.70% w/w,about 0.55% w/w-0.75% w/w, about 0.60% w/w-0.65% w/w, about 0.60%w/w-0.70% w/w, about 0.60% w/w-0.75% w/w, about 0.65% w/w-0.70% w/w,about 0.65% w/w-0.75% w/w, or about 0.70% w/w0.75% w/w. In some furtherembodiments the concentration of the non-ionic non-fluorous surfactantcan be adjusted to optimize the droplet stability and droplet sizewithout inhibiting the PCR assay.

In some embodiments, the concentration of fluorosurfactant is in a rangeof about 1.0-6.0 mM and the concentration of non-ionicnon-fluorosurfactant is in a range of about 0.1%-3.0% weight percent. Ina further embodiment, the fluorosurfactant has a structure of Formula Iand a concentration of about 2.5 mM, and the non-ionicnon-fluorosurfactant is Pluronic® F-98 and has a concentration of about0.5% w/w-1.5% w/w. In some cases, the fluorosurfactant has a structureof Formula I and a concentration of about 2.5 mM, and the non-ionicnon-fluorosurfactant is Pluronic® F-98 and has a concentration of about0.50% w/w-0.60% w/w, about 0.50% w/w-0.65% w/w, about 0.50% w/w-0.70%w/w, about 0.55% w/w-0.60% w/w, about 0.55% w/w-0.65% w/w, about 0.55%w/w-0.70% w/w, about 0.55% w/w-0.75% w/w, about 0.60% w/w-0.65% w/w,about 0.60% w/w-0.70% w/w, about 0.60% w/w-0.75% w/w, about 0.65%w/w-0.70% w/w, about 0.65% w/w-0.75% w/w, or about 0.70% w/w0.75% w/w.

In the methods of the present invention, the non-ionic non-fluoroussurfactants reside essentially in the aqueous phase. In some cases atleast about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% of the non-ionicnon-fluorous surfactant resides in the aqueous phase of the aqueousdroplets.

Intercalating Dye

Intercalating dyes are generally aromatic cations with planar structuresthat insert between stacked base pairs in the DNA duplex, an arrangementthat provides an environmentally dependent fluorescence enhancement fordye molecules and creates a large increase in the fluorescence signalrelative to the free dye in solution. The signal enhancement provides aproportional response, allowing direct quantitative DNA measurements.Preferred intercalating dyes in the present disclosure includefluorescent dyes. The dye can be a cyanine or a non-cyanineintercalating die. In some cases, the intercalating dye is a cyaninedye. In some cases the cyanine dye can be Thiazole Orange, SYBR® (e.g.Sybr Green I, Sybr Green II, Sybr Gold, SYBR DX), Oil Green, CyQuant GR,SYTOX Green, SYTO9, SYT010, SYTO17, SYBR14, Oxazile Yellow, ThiazoneOrange, SYTO, TOTO, YOYO, BOBO, and POPO. In some cases the dye is anon-cyanine dye. In some cases the non cyanine dye is pentacene,anthracene, naphthalene, ferrocene, methyl viologen, tri-morpholinoammonium, propidium (e.g., propidium iodide) or another aromatic orheteroaromatic derivative. In some cases, the intercalating dye can beEvaGreen®.

Other Additives

In some embodiments, an amplification reaction can also comprise one ormore additives including, but not limited to, non-specificbackground/blocking nucleic acids (e.g., salmon sperm DNA),biopreservatives (e.g., sodium azide), PCR enhancers (e.g. Betaine,Trehalose, etc.), and inhibitors (e.g., RNAse inhibitors). The one ormore additives can include, e.g., 2-pyrrolidone, acetamide,N-methylpyrolidone (NMP), B-hydroxyethylpyrrolidone (HEP), propionamide,NN-dimethylacetamide (DMA), N-methylformamide (MMP),NN-dimethylformamide (DMF), formamide, N-methylacetamide (MMA), dimethylsulfoxide (DMSO), polyethylene glycol, betaine, tetramethylammoniumchloride (TMAC), 7-deaza-2′-deoxyguanosine, bovine serum albumin (BSA),T4 gene 32 protein, or glycerol.

Devices and Systems

In another aspect, the present disclosure provides a droplet generationsystem, comprising: (a) a first channel in fluid communication with acarrier fluid source, wherein the carrier fluid source comprises an oiland a fluorosurfactant, and wherein the fluorosurfactant is selectedfrom Formula I, II and/or III; (b) a second channel in fluidcommunication with a sample source, wherein the sample source comprisesa target nucleic acid, a non-ionic surfactant and an intercalating dye;(c) a droplet channel; and (d) a droplet source; wherein the firstchannel meets the second channel at an intersection; the intersectionreceives a sample from the sample source and a carrier fluid from thecarrier fluid source and generates emulsified droplets; and theemulsified droplets flow along the droplet channel to the dropletsource.

FIG. 7 shows a schematic representation of a droplet generation system100 comprising a sample source 105 in fluid communication with a samplechannel 110, and a carrier fluid source 115 in fluid communication witha carrier fluid channel. The sample channel 110 and the carrier fluidchannel 115 meet at an intersection or a droplet generation point 125.During operation the carrier fluid comprising oil and a fluorosurfactantfrom the carrier fluid source 115 is directed through the carrier fluidchannels 120 to the intersection 125. The sample from the sample source105 comprising a target nucleic acid, a non-ionic surfactant and anintercalating dye is also directed to the intersection through thesample channels, wherein a sample partition generate a dropletcomprising an aqueous phase in an oil phase. A droplet thus formed flowsin a droplet channel 130 from the intersection 125 to a droplet source135 for holding the droplets.

In some situations, the system includes a detection assembly in fluidcommunication with the fluid flow path. The detection assembly may besituated along at least a portion of the detection channel between theintersection and the collection source. The detection assembly can beconfigured to detect signals from droplets in the fluid flow path, suchas upon flowing through the detection channel. The detection assemblycan include an optical sensor or other electronic detector that issensitive to a select frequency of light. The sensor can be adapted todetect fluorescent emission, for example. In some cases, the detectionassembly can include an excitation source, such as a light source thatis adapted to induce fluorescence in the fluid. One or more opticalelements (e.g., mirrors, lenses) can be provided to direct light emittedfrom the fluid to the detection assembly, and/or to direct light from alight source to the fluid.

In some embodiments, the system includes a pressure source forfacilitating the flow of droplets from the intersection to thecollection source. The pressure source can be a source of positivepressure operatively coupled to the carrier fluid and/or sample source,or a source of negative pressure (i.e., vacuum) operatively coupled tothe fluid flow path, such as by way of the collection source. The sourceof negative pressure can be a pumping system.

The droplet generation system 100 can include a droplet detector. Insome examples, the droplet detector is fluidically isolated from thedroplet channel 130, but upon formation, droplets are transferred to thedroplet detector for droplet detection and sample quantification. Inother examples, the droplet detector is in fluid communication with thedroplet channel 130. For instance, the droplet detector can be situatedalong the droplet channel 130. Various features, components and uses ofthe droplet detector may be as described in, for example, U.S. PatentPublication No. 2010/0173394 to Colston et al. (“Droplet-based assaysystem”), which is entirely incorporated herein by reference.

Method of Nucleic Acid Detection

Another aspect of the disclosure provides a method of detecting anucleic acid. The method comprises: (a) providing an oil compositioncomprising a fluorosurfactant, wherein the fluorosurfactant is selectedfrom the group consisting of Formula I, II and III; (b) providing anaqueous composition comprising a target nucleic acid, a non-ionicsurfactant and an intercalating dye; (c) contacting the oil compositionof (a) with the aqueous composition of (b), thereby generating aplurality of droplets suspended in a continuous phase; (d) thermallycycling the plurality of droplets to amplify the target nucleic acid;and (e) detecting the target nucleic acid.

The plurality of droplets generated by this method may be aqueousdroplets encapsulated by an oil phase. The encapsulation may provideenhanced stability of the droplets and/or dye concentration inside thedroplets.

In some embodiments, less than about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, orabout 50% of total intercalating dyes are detected outside the aqueousphase of the aqueous droplets. In some cases, the detection can be afterabout 5 thermal cycles, about 10 thermal cycles, about 15 thermalcycles, about 20 thermal cycles, about 30 thermal cycles, about 40thermal cycles, about 50 thermal cycles, about 60 thermal cycles, about70 thermal cycles, about 80 thermal cycles, about 90 thermal cycles,about 100 thermal cycles, about 120 thermal cycles, about 150 thermalcycles, about 200 thermal cycles, about 300 thermal cycles, about 400thermal cycles, about 500 thermal cycles or even more thermal cycles.

In some embodiments, less than about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, orabout 50% of a specific intercalating dye (e.g. EvaGreen®, SYBR Green®)can be detected outside the aqueous phase of the aqueous droplets. Insome cases, the detection can be after about 5 thermal cycles, about 10thermal cycles, about 15 thermal cycles, about 20 thermal cycles, about30 thermal cycles, about 40 thermal cycles, about 50 thermal cycles,about 60 thermal cycles, about 70 thermal cycles, about 80 thermalcycles, about 90 thermal cycles, about 100 thermal cycles, about 120thermal cycles, about 150 thermal cycles, about 200 thermal cycles,about 300 thermal cycles, about 400 thermal cycles, about 500 thermalcycles or even more thermal cycles.

Individually, each of the plurality of droplets may comprise 0 targetnucleic acids, 1 target nucleic acid, 2 target nucleic acids, 3 targetnucleic acids, 4 target nucleic acids, 5 target nucleic acids, 6 targetnucleic acids, 7 target nucleic acids, 8 target nucleic acids, 9 targetnucleic acids, 10 target nucleic acids, 11 target nucleic acids, 12target nucleic acids, 13 target nucleic acids, 14 target nucleic acids,or 15 target nucleic acids prior to thermal cycling. After thermalcycling, each of plurality of droplet can comprise about 15×10⁴⁰ targetnucleic acids, about 14×10⁴⁰ target nucleic acids, about 13×10⁴⁰ targetnucleic acids, about 12×10⁴⁰ target nucleic acids, about 11×10⁴⁰ targetnucleic acids, about 10×10⁴⁰ target nucleic acids' about 9×10⁴⁰ targetnucleic acids, about 8×10⁴⁰ target nucleic acids, about 7×10⁴⁰ targetnucleic acids, about 6×10⁴⁰ target nucleic acids, about 5×10⁴⁰ targetnucleic acids, about 4×10⁴⁰ target nucleic acids, about 3×10⁴⁰ targetnucleic acids, about 2×10⁴⁰ target nucleic acids, about 1×10⁴⁰ targetnucleic acid or 0 target nucleic acids.

The average number of target nucleic acid copies present per dropletprior to thermal cycling can be in the range of about 0.00005-5 targetnucleic acid per droplet. In some cases the average number of targetnucleic acids present per droplet prior to thermal cycling is about0.0001-5 target nucleic acids per droplet, about 0.001-5 target nucleicacids per droplet, about 0.01-5 target nucleic acids per droplet, about0.1-5 target nucleic acids per droplet or about 1-5 target nucleic acidsper droplet. Furthermore, after thermal cycling the average number oftarget nucleic per droplet could be less than about 5.5×10⁶ targetnucleic acids per droplet, about 5.5×10⁷ target nucleic acids perdroplet, about 5.5×10⁸ target nucleic acids per droplet, about 5.5×10⁹target nucleic acids per droplet, 5.5×10¹¹ target nucleic acids perdroplet, or about 5.5×10¹² target nucleic acids per droplet. In someembodiments, the detecting comprises measuring fluorescence from theintercalating dyes.

PCR nucleic acid amplification may rely on thermal cycling (i.e.,alternating cycles of heating and cooling) to achieve successive roundsof replication. In an example, during DNA melting, two strands in a DNAdouble helix are separated (e.g., at a high temperature). Each strandcan then be used (e.g., at a lower temperature) as the template in DNAsynthesis by the DNA polymerase to selectively amplify the target DNA.

PCR can be performed by thermal cycling between two or more temperatureset points, such as a higher melting (denaturation) temperature and alower annealing/extension temperature, or among three or moretemperature set points, such as a higher melting temperature, a lowerannealing temperature, and an intermediate extension temperature, amongothers. PCR can be performed with a thermostable polymerase. Somenon-limiting examples of thermostable DNA polymerase include Taq DNApolymerase, Pfu DNA polymerase, Vent polymerase, S-Tbr polymerase, Tthpolymerase, or a combination thereof.

Detection of PCR products can be accomplished using fluorescencetechniques. DNA-intercalating dyes exhibit an increased fluorescenceupon binding to DNA and can provide a quantitative readout of the amountof DNA present in a reaction volume. As this amount of DNA increasesover the course of a reaction, the fluorescence intensity increases.Fluorescence detection can be achieved using a variety of detectordevices equipped with a module to generate excitation light that can beabsorbed by a fluorescent dye, as well as a module to detect lightemitted by the fluorescent dye. In some embodiments, droplets can bedetected in bulk. For example, samples can be allocated in tubes thatare placed in a detector that measures bulk fluorescence from tubes. Insome embodiments, one or more droplets can be partitioned into one ormore wells of a plate, such as a 96-well plate, and fluorescence ofindividual wells can be detected using a fluorescence plate reader.

EXAMPLES Example 1

FIG. 1 and FIG. 2 show that the dye leaching response is dependent onthe concentration of the carboxylic acid XI.

1 ml aliquots of an aqueous solution of SYBR Green (finalconcentration=0.5×) were overlaid on 1 ml solutions of HFE-7500containing known concentrations of carboxylic acid XI (0.01 mM-0.10 mM).At 1 minute intervals a 1 μl aliquot of the aqueous SYBR Green solutionwas taken for interrogation by UV-Vis spectroscopy at 528 nm using aNanoDrop UV/Vis spectrophotometer (Thermo Scientific). The UV-Visresponse was normalized then plotted as a function of time (minutes) foreach concentration. FIG. 1 clearly indicates that the rate of leachingof SYBR Green from the aqueous phase into the fluorous phase isproportional to the concentration of carboxylic acid XI present in thefluorinated oil (HFE-7500). The rate of dye leaching increases as theconcentration of carboxylic acid XI increases.

A second experiment was performed in an analogous fashion to thatdescribed above. In this case, the first solution contained carboxylicacid XI at a concentration of 2.5 mM in HFE-7500, the second solutioncontained the fluorosurfactant XII at a concentration of 2.5 mM inHFE-7500, while the third solution was neat HFE-7500 (used as acontrol). FIG. 2 clearly illustrates retention of SYBR Green dye withinthe aqueous phase for neat HFE-7500 and the solution containing 2.5 mMof fluorosurfactant XII. The relative UV-Vis responses remained constantover time in both cases, this was in stark contrast to the HFE-7500solution containing 2.5 mM carboxylic acid XI which significantlydecreased over time.

Example 2

FIG. 4 shows that Bis-Krytox 157FSH-PEG1000 (XII) provides stabilizationof aqueous-in-fluorous oil droplets over a large range ofconcentrations, and in conjunction with the aqueous surfactant(Pluronic®-F98) the optimum concentration is 2.5 mM.

In this experiment the concentration range of fluorosurfactant XIIrequired for adequate droplet stabilization was studied throughfunctional testing. Various solutions of fluorosurfactant XII, rangingin concentration from 1.0×10⁻⁵-0.1 M, were prepared in HFE-7500. Assaydroplets containing 1 cpd. S. Aureus template, primers, DNA polymerase,and 1× EvaGreen® in a buffered aqueous ddPCR master mix were generatedfrom each of the fluorosurfactant solutions (4 wells, or reactions, perconcentration) by a standard method using a QX100™ Droplet Generator(Bio-Rad). Droplets were transferred to a 96-well PCR plate then cycledon a C1000 Touch™ Thermal Cycler (Bio-Rad) using a standard ddPCRthermal cycling protocol. Each well of droplets was then interrogatedusing a modified QX100™ Droplet Reader to count positive (highfluorescence intensity) and negative (low fluorescence intensity)droplets. FIG. 4 indicates a wide working concentration range forfluorosurfactant XII of 1.0×10⁻²-2.5×10⁻⁵ M in HFE-7500. Atconcentrations below or above these values droplet integrity suffers asevidenced by the large increase in ‘rain’ (off-amplitude signals) andmerging of the positive and negative droplet populations.

Example 3

FIG. 5 shows the effect of Pluronic® type and concentration on dropletstability. In this experiment the droplet stabilizing effect ofPluronic® was studied by adding various types of Pluronic® to theaqueous phase of the droplets at a range of concentrations. The types ofPluronic® studied included F-38, F-68, and F-98, typical concentrationswere 0.5-5.0% w/w. Buffered aqueous solutions containing 1× EvaGreen®,primers, and the specified concentration and type of Pluronic® wereprepared. Droplets containing the various aqueous solutions weregenerated using fluorosurfactant XII at a concentration of 2.5 mM inHFE-7500 (4 wells per Pluronic® type and concentration) by a standardmethod using a QX100™ Droplet Generator (Bio-Rad). Droplets weretransferred to a 96-well PCR plate then cycled on a C1000 Touch™ ThermalCycler (Bio-Rad) using a standard ddPCR thermal cycling protocol. Eachwell of droplets was then interrogated using a modified QX100™ DropletReader to assess the droplet integrity using various droplet qualitymetrics. FIG. 5 suggests that as the MW of the Pluronic® increases(while maintaining a constant HLB), from Pluronic® F-38 to F-98 thestability of the droplets to thermal degradation/coalescence alsoincreases.

Example 4

FIG. 7 shows the effect of Pluronic®-F98 concentration on droplet size.

This experiment involved the titration of Pluronic® F-98 in an aqueousmaster mix from 0.50-1.50% w/w to determine the effect of Pluronic®concentration on the measured droplet size following droplet generation.Buffered aqueous solutions containing 1× EvaGreen®, primers, and thespecified concentration of Pluronic® were prepared. Droplets containingthe various aqueous solutions were generated using fluorosurfactant XIIat a concentration of 2.5 mM in HFE-7500 (8 wells per Pluronicconcentration) by a standard method using a QX100™ Droplet Generator(Bio-Rad). The droplets generated were removed from the outlet well,then sized by microscopy on a glass microscope slide. FIG. 7 clearlyindicates that as the concentration of Pluronic® F-98 increase the meanradius of the droplets generated decreases.

Example 5

FIGS. 8A and 8B show the use of EvaGreen® dye and micro-emulsion(droplets) comprising of the combination of continuous phase (2.5 mMBis-Krytox 157FS_(H)-PEG₁₀₀₀, XII) and aqueous phase (050-0.75% w/wPluronic®-F98, buffered, pH—8.3-8.4) in ddPCR assay.

This experiment involved performing a ddPCR assay using EvaGreen® dyeunder the optimized surfactant conditions (2.5 mM fluorosurfactant XIIin HFE-7500, 0.50% w/w Pluronic® F-98 in buffered aqueous ddPCR mastermix). Assay droplets (48 wells/reactions) containing 1 cpd. S. Aureustemplate, primers, DNA polymerase, 1× EvaGreen®, and 0.50% w/w Pluronic®F-98, in a buffered aqueous ddPCR master mix were generated using theoptimized droplet generation oil (2.5 mM fluorosurfactant XII inHFE-7500) by a standard method using a QX100™ Droplet Generator(Bio-Rad). Droplets were transferred to a 96-well PCR plate then cycledon a C1000 Touch™ Thermal Cycler (Bio-Rad) using a standard ddPCRthermal cycling protocol. Each well of droplets was then interrogatedusing a modified QX100™ Droplet Reader to count positive (highfluorescence intensity) and negative (low fluorescence intensity)droplets. FIG. 8A displays the ddPCR data for a single well/reaction,showing clear separation between the positive and negative droplets.FIG. 8B displays the ddPCR data for the 48 wells/reactions combined,again, illustrating distinct positive and negative bands. In this caseit is also clear that the intercalating dye is not leaching, as thepositive amplitude remains constant over the time taken for dropletinterrogation (˜1.5 hours).

Example 6

Scheme 4 shows an example of a synthetic route for the preparation offluorosurfactant I.

Solvent Preparation Toluene

Toluene (125 ml) was dried over CaH₂ and solid Na flakes for at least 24hours then freshly distilled prior to use. Benzophenone was added to thetoluene during drying to act as an indicator, the toluene was a dark,vibrant, blue/purple color before distillation. Alternatively, toluenewas dispensed directly from an anhydrous solvent dispenser if available.

HFE-7100

HFE-7100 (500 ml) was to be dried over CaH₂ for at least 24 hours thenfreshly distilled prior to use. Alternatively, HFE-7100 may be dispenseddirectly from an anhydrous solvent dispenser if available.

Tetrahydrofuran (THF)

THF (125 ml) was dried over CaH₂ and solid Na flakes for at least 24hours then freshly distilled prior to use. Benzophenone was added to theTHF during drying to act as an indicator, the THF must be a dark,vibrant, blue/purple color before distillation. Alternatively, THF maybe dispensed directly from an anhydrous solvent dispenser if available.

Reagent Preparation

The MW of each new carboxylic acid XI lot number was determined by ¹⁹FNMR prior to use. PEG(1000) was dissolved in dry toluene (125 ml) thenevaporated to dryness under reduced pressure (<10 mbar) at 60° C. toremove any traces of H₂O by azeotropic distillation (immediately beforeuse). Oxalyl Chloride, Dimethylformamide and Triethylamine were used assupplied by Sigma-Aldrich. To ensure the anhydrous integrity of oxalylchloride and triethylamine were both purchased fresh prior to use. Thereagents were not used for manufacturing if they had been opened formore than a month prior to use.

Synthetic Method

Carboxylic acid XI (300.0 gram (g), 49.63 millimoles (mmol)) anddimethylformamide (0.192 milliliter (ml), 2.48 mmol) were added to a1000 ml round bottom flask (RBF) equipped with a magnetic stirring bar.The RBF was purged with N₂ (or Ar), stoppered with a rubber septum,topped with a N₂ (or Ar) filled balloon, and then freshly distilledHFE-7100 (250 ml) was added by syringe. A reflux condenser topped with arubber septum and N₂ (or Ar) filled balloon was attached to the RBF,then the contents were heated (sand bath set at 90° C.) with stirring(˜650 revolutions per minute (rpm)) for 5 minutes before the slowaddition of oxalyl chloride (21 ml, 245 mmol) by syringe. The clearsolution turned a clear, light yellow color, and a large amount of gaswas produced (CAUTION!), excess pressure was relieved with an uncappedneedle (19 gauge), and manual venting of the rubber septum if required.Following stirring for 10 minutes at reflux, the solution frothed again,produced a large amount of gas, and became a milky white color.Following a further 5 minutes stirring at reflux the solution returnedto an opaque, light yellow color. After an additional 15 minutesstirring at reflux, the solution had become a clear, light yellow color.

This solution was stirred at reflux (60° C.) for a total of 4 hours,during this time the solution became a clear orange color and containeda small amount of suspended solid brown particulate. The solution wasallowed to cool to room temperature, and then excess solvent and oxalylchloride were removed under reduced pressure (<10 mbar) at 60° C. togive the crude acid chloride as an orange oil containing suspended brownparticulate.

Freshly distilled HFE-7100 (250 ml) was added to the crude acid chloridein the 1000 ml RBF (which was capped with a rubber septum and N₂ (or Ar)filled balloon) by syringe. This solution was then refluxed withstirring (˜650 rpm) at 60° C. (sand bath set at 90° C.) under N₂ (or Ar)for 5 minutes, before addition of a solution of PEG (24.8 g, 24.8 mmol)and triethylamine (14 ml, 100 mmol) in dry THF (125 ml) by syringe (ordouble-tipped needle, or an appropriate transfer line).

Note: The reaction mixture immediately fumed and turned a milky whitecolor due to a large amount of white precipitate (NEt₃.HCl) forming.

This reaction mixture was refluxed at 66° C. (sand bath set at 90° C.)under N₂ (or Ar) with stirring (˜650 rpm) for approximately 18 hours,during this time the reaction mixture became a tan color and a smallamount of brown/black precipitate adhered to the walls of the RBF. Thereaction mixture was allowed to cool to RT, then excess solvent andtriethylamine were removed under reduced pressure (<10 mbar) at 60° C.to give the crude Formula I as an orange/tan semi-solid. The crudeproduct was dissolved in HFE-7100 (1750 ml), then added to a 2000 mlseparating funnel, this solution was allowed to settle overnight (thesolid impurities floated to the top of the HFE-7100). The fluorous phasewas slowly filtered through a fitted Buchner funnel (porosity 4-8 μm)under vacuum in portions of ˜500 ml and collected in a 1000 mlpear-shaped evaporating flask. The solvent was evaporated under reducedpressure at 60° C. following each collection. The remaining light tansolid residue present in the 2000 ml separating funnel was washed withadditional HFE-7100 (250 ml). This solution was allowed to settleovernight. The solution was then slowly filtered through a frittedBuchner funnel (porosity 4-8 μm) under vacuum in portions of ˜500 ml andcollected in a 1000 ml pear-shaped evaporating flask. The solvent wasevaporated under reduced pressure at 60° C. following each collection.The residue was combined with the previous fluorous extracts, thenevaporated to dryness under reduced pressure (<10 mbar) at 70° C. toyield Formula I (323.9 g, 25.02 mmol) as an extremely viscousorange/brown syrup.

Example 7 Characterization of the Fluorosurfactant XII NMR Spectroscopyof Carboxylic Acid XI

A ¹⁹F NMR spectrum of the specific lot of starting carboxylic acid XIused for synthesis was collected ‘neat’ at 50° C. with a total of 128scans. The typical ¹⁹F NMR spectrum for the carboxylic acid is shown inFIG. 9. The actual average molecular weight (MW) for carboxylic acid XIwas calculated based on the ¹⁹F NMR spectra. The average MW calculatedshould be within 5500-6500 atomic mass units (amu).

An example of MW calculation from ¹⁹F NMR spectrum is shown in FIG. 12.The CF ₂—CF₃ signal was ‘easily’ identified and well resolved withreliable integration (˜131 ppm). To calculate the MW range, the integralof the CF ₂—CF₃ (˜131 ppm) signal was compared to the integral of thesignals from the repeating monomer units (˜145 ppm and ˜1 ppm) todetermine n.

The three integrals were normalized so that the CF ₂—CF₃ signal wasequal to 2.00:

-   -   So: 1.11×1.80=2.00, 18.43×1.80=33.21, and 100.00×1.80=180.18    -   So: n=[Int (CF—CF₂)/1]/[Int (CF ₂—CF₃)/2] e.g.        n=(33.21/1)/(2.00/2)=33.21        -   OR    -   n=[Int ((CF ₂—CF+CF ₃—CF—CF₂)-2)/5]/[Int (CF ₂—CF₃)/2]        e.g. n=((180.18-2)/5)/(2.00/2)=35.64        Once ‘n’ is determined, MW's of repeating monomer and terminal        units were added together:

$\mspace{20mu} \begin{matrix}{{MW}_{({{carboxylic}\mspace{14mu} {acid}\mspace{14mu} {XI}})} = {{{MW}( {C_{2}F_{4}{COOH}} )} + \lbrack {n \times {{MW}( {C_{3}F_{6}O} )}} \rbrack +}} \\{{{MW}( {C_{3}F_{7}O} )}} \\{= {145.03 + \lbrack {n \times (166.02)} \rbrack + 185.02}}\end{matrix}$ $\mspace{20mu} \begin{matrix}{{{So}\text{:}\mspace{14mu} {MW}_{({{carboxylic}\mspace{14mu} {acid}\mspace{14mu} {XI}})}} = {145.03 + ( {33.21 \times 166.02} ) + 185.02}} \\{= 5843.57} \\{= {145.03 + ( {35.64 \times 166.02} ) + 185.02}} \\{= 6247.00}\end{matrix}$ $\mspace{20mu} \underset{\_}{OR}$So:  Average  MW(_((carboxylic  acid  XI)) = (5843.57 + 6247.00)/2 = 6045.29

NMR Spectroscopy of Fluorosurfactant XII

The sample of fluorosurfactant XII for ¹⁹F and ¹³C NMR analysis wasprepared by dissolving 1 g of the product in 0.2 ml of hexafluorobenzene(C₆F₆). Approximately 0.7 ml of this solution was transferred to astandard NMR tube (5 mm×180 mm) to fill the tube to a depth ofapproximately 5 cm. The ¹⁹F spectrum was collected ‘neat’ at 50° C. witha total of 128 scans, the ¹³C spectrum is collected ‘neat’ at 50° C.scanning continuously for about 2 hours.

Typical ¹⁹F and ¹³C spectra of fluorosurfactant XII are shown in FIGS.13 and 14 respectively. Of particular interest in the ¹³C NMR spectra offluorosurfactant XII is the resonance due to the carbonyl carbon (C=0)at about 157 ppm. This resonance has undergone a significant up-fieldshift when compared to the resonance of the corresponding carbonylcarbon at about 162 ppm in the ¹³C NMR spectra of the carboxylic acidstarting material XI (FIG. 10).

In the ¹³C NMR spectrum of high purity fluorosurfactant XII, arelatively weak resonance due to the carbonyl carbons (C=0) of the twoester linkages was present at 157±2 ppm, while the resonance due to thecarbonyl carbon (COOH) of the carboxylic acid XI observed at 162±2 ppmwas absent.

ATR-FTIR

This qualitative technique was used to confirm the conversion of thecarbonyl carbon of the carboxylic acid XI starting material to the twoester linkages formed in the fluorosurfactant XII. This analysis wasperformed using the Bruker Tensor 27 IR spectrophotometer with thePlatinum ATR module installed. Following collection of the backgroundspectrum, a small drop of the fluorosurfactant XII sample was smearedacross the ATR crystal and the spectrum of the sample was collected withthe following parameters; resolution=4, number of scans=32, andwavelength range=4000 to 500 cm⁻¹. A peak correlating to the C=0 stretchwas found to be present with the maximum occurring at about 1784±6 cm⁻¹indicating the conversion of the carboxylic acid XI to the desiredfluorosurfactant XII.

Fluorescence

A custom quantitative technique was developed in house to determine theremaining amount of carboxylic acid XI starting material left in thefluorosurfactant XII product as an impurity, and to determine thepercentage purity of the fluorosurfactant XII.

A 100 ml of a 1.000 mM stock solution of the carboxylic acid XI wasprepared in a 100 ml volumetric flask. Nine subsequent standards wereprepared from stock solutions according to the volumes indicated in theTable 1 below (final concentration range of 1.000 mM to 0.010 mM). 25 mlof a 10× solution of SYBR green in deionized H₂O was also prepared in a25 ml volumetric flask.

The amount of carboxylic acid XI required for 1.000 mM stock wasdetermined by the following formula

Mass (carboxylic acid XI) in grams=0.001 M×0.1 L×Actual MW of carboxylicacid XI as determined by ¹⁹F NMR analysis.

TABLE 1 The prepared stock solutions of carboxylic acid XI. RequiredRequired Required Desired stock concentration of volume of volume Finalconcentration stock stock HFE-7500 volume (mM) (mM) (ml) (ml) (ml) 1.000(stock) — — 100.0 100.0 0.750 1.000 7.5 2.5 10.0 0.500 1.000 5.0 5.010.0 0.400 1.000 4.0 6.0 10.0 0.300 1.000 3.0 7.0 10.0 0.200 1.000 2.08.0 10.0 0.100 1.000 5.0 45.0 50.0 0.075 0.100 7.5 2.5 10.0 0.050 0.1005.0 5.0 10.0 0.010 0.100 1.0 9.0 10.0

A Varian Cary Eclipse spectrofluorimeter instrument was used for thepresent analysis and the temperature was set to 20° C. 2 ml of thecarboxylic acid XI 0.010 M stock solution was transferred to clean 3 mLquartz cuvette, on which was carefully overlaid 1 ml of the SYBR 10×stock solution. The fluorescence spectra of the fluorous phase of thissample was then collected at ˜542 nm over a period of time (˜1 hour) at20° C. using a Varian Cary Eclipse spectrofluorimeter (AgilentTechnologies, Inc.).

The above steps were repeated for the additional nine standards preparedand for the 2.5 mM solution fluorosurfactant XII containing an unknownamount of acid XI as purity. Between each run, the quartz cuvette wasthoroughly rinsed with 70% EtOH solution and HFE-7500 and was driedusing compressed air.

The kinetic leaching profiles of each of the above solutions were fittedto the equation 1 shown below to determine the pseudo-first orderkinetic rate constant (k) for each of the standards and the unknownfluorosurfactant XII sample (FIG. 16A). A plot of k versus concentrationfor the standards was obtained to construct a calibration curve shown inFIG. 16B.

[B]=[B] ₀ e ^(−k[A]ot)  Equation 1:

where:

-   -   [B]=fluorescence intensity    -   [B]₀=fluorescence intensity at t=0    -   e=natural log    -   k=rate constant    -   [A]₀=1    -   t=time

The unknown concentration of carboxylic acid XI in the 2.5 mMfluorosurfactant XII sample was determined from the calibration curve.For example in FIG. 16B, the pseudo first kinetic rate constant of7.25×10⁻⁴ sec⁻¹ corresponds to about 0.42 mM concentration of thecarboxylic acid XI. The purity of the synthesized fluorosurfactant wasdetermined using the formulas below:

Theoretical  mass  of  fluorosurfactant  in  1  L  of  2.5  mM  XII = concentration  (M) × volume  (L) × MW  (XII) = 0.0025  (M) × 1  (L) × 13054 = 32.64  gActual  mass  of  carboxylic  acid  XI  in  1  L  of  2.5  mM  XII:concentration  (M)  (from  the  calibration  curve) × volume  (L) × MW  (XI) = (0.42  (mM)/1000) × 1  (L) × 6045 = 2.54  gActual  mass  of  fluorosurfactant  in  1  L  of  2.5  mM  XII = Theoretical  mass  fluorosurfactant  XII  (g) − Calculated  mass  of  carboxylic  acid  XI  (g) = 32.64  (g) − 2.54  (g) = 30.10  (g)${{Purity}\mspace{14mu} {of}\mspace{14mu} {fluorosurfactant}\mspace{14mu} {XII}\mspace{14mu} ( {\% \mspace{14mu} w\text{/}w} )} = {{\frac{{Actual}\mspace{14mu} {mass}\mspace{14mu} {fluorosurfactant}\mspace{14mu} {XII}\mspace{14mu} (g)}{{Theoretical}\mspace{14mu} {mass}\mspace{14mu} {fluorosurfactant}\mspace{14mu} {XII}\mspace{11mu} (g)} \times \frac{100}{1}} = {{( {30.10\mspace{14mu} (g)\text{/}32.64\mspace{11mu} (g)} ) \times ( {100\text{/}1} )} = {92.2\% \mspace{14mu} w\text{/}w}}}$

Elemental Analysis

Analysis was carried out for the elements C, H and F and each analysiswas performed in duplicate. The theoretical molecular formula andpercentage composition of the elements C, H and F were calculated basedupon the average molecular weights of the starting materials. Theelemental results were recorded and compared with the theoreticalelemental composition. The difference between the theoretical elementalcomposition and actual elemental analysis results was less than 0.5%. Arepresentative data from the elemental analysis of fluorosurfactant XII,average molecular formula=C₂₆₀H₉₀O₉₅F₄₃₀, is tabulated in Table 2.

TABLE 2 Elemental analysis of fluorosurfactant XII. ElementFluorosurfactant X11 (theoretical) Analysis 1 Δ Analysis 2 Δ C - 24.20%24.12% 0.08% 24.13% 0.07% H - 0.70%  0.58% 0.12%  0.52% 0.18% O - 11.78%— — — — F - 63.31% 63.47% 0.16% 63.62% 0.31% Note: O content is unableto be determined if F content exceeds 30%

Example 8. Analysis of Fluorosurfactant Mixtures

FIG. 17 shows the analysis of EvaGreen® dye and micro-emulsion(droplets) comprising the various fluorosurfactant mixtures in a ddPCRassay.

This experiment involved performing a ddPCR assay using EvaGreen® dyeunder the various surfactant conditions, wherein: A=Formula I; B=FormulaI+5 mol % Formula XI+5 mol % Formula II; C=Formula I+10 mol % FormulaXI+10 mol % Formula II; D=Formula I+20 mol % Formula XI+20 mol % FormulaII; E=Formula I+50 mol % Formula XI+50 mol % Formula II. Assay droplets(96 nwells/reactions) containing 1 cpd. S. Aureus template, primers, DNApolymerase, 1× EvaGreen, and 0.50% w/w Pluronic® F-98, in a bufferedaqueous ddPCR master mix were generated using the optimized dropletgeneration oil (2.5 mM fluorosurfactant XII in HFE-7500) by a standardmethod using a QX200™ Droplet Generator (Bio-Rad). Droplets weretransferred to a 96-well PCR plate then cycled on a C1000 Touch™ ThermalCycler (Bio-Rad) using a standard ddPCR thermal cycling protocol. Eachwell of droplets was then interrogated using a QX200™ Droplet Reader tocount positive (high fluorescence intensity) and negative (lowfluorescence intensity) droplets. FIG. 17 displays the ddPCR data forthe 96 wells/reactions combined, illustrating distinct positive andnegative bands. The spikes seen in window A indicate large extra-clusterdroplets, which are reduced upon addition of various amounts of formulaII and formula XI.

While exemplary examples and embodiments of the present invention havebeen shown and described herein, it will be obvious to those skilled inthe art that such examples and embodiments are provided by way ofexample only. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the invention.It should be understood that various alternatives to the embodiments ofthe invention described herein may be employed in practicing theinvention. It is intended that the following claims define the scope ofthe invention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

We claim:
 1. A composition, comprising: a continuous phase including anoil and a fluorosurfactant mixture, wherein said fluorosurfactantmixture includes a triblock copolymer comprising a first polyethyleneglycol (PEG) block covalently linked to a respectivepolyhexafluoropropylene oxide (PFPE) block at each end of the first PEGblock, a diblock copolymer comprising a second PEG block covalentlylinked to a PFPE block at only one end of the second PEG block, and aPFPE carboxylic acid; and aqueous droplets suspended in the continuousphase and comprising nucleic acid, a non-ionic non-fluorosurfactant, andan intercalating dye; wherein the first PEG block and each respectivePFPE block of the triblock copolymer are linked to one another by anester bond, and wherein the second PEG block and the PFPE block of thediblock copolymer are linked to one another by an ester bond.
 2. Thecomposition of claim 1, wherein the triblock copolymer has the formula

wherein each of m₁ and m₂ is a number from about 10 to 100, and whereinn is a number from about 15 to
 30. 3. The composition of claim 2,wherein each of m₁ and m₂ is a number from about 20 to 40, and wherein nis a number from about 10 to
 60. 4. The composition of claim 2, whereinthe diblock copolymer has the formula

wherein m₃ is a number from about 20 to 50, wherein n₂ is a number fromabout 8 to 30, and wherein R is H or an alkyl group.
 5. The compositionof claim 4, wherein the PFPE carboxylic acid has the formula

wherein n is about 15 to
 50. 6. The composition of claim 2, wherein thePFPE carboxylic acid has the formula

wherein n is about 15 to
 50. 7. The composition of claim 1, wherein thediblock copolymer has the formula

wherein m₃ is a number from about 20 to 50, wherein n₂ is a number fromabout 8 to 30, and wherein R is H or an alkyl group.
 8. The compositionof claim 7, wherein the PFPE carboxylic acid has the formula

wherein n is about 15 to
 50. 9. The composition of claim 1, wherein thePFPE carboxylic acid has the formula

wherein n is about 15 to
 50. 10. The composition of claim 1, wherein thenon-ionic non-fluorosurfactant is a copolymer of ethylene oxide andpropylene oxide.
 11. The composition of claim 10, wherein the non-ionicnon-fluorosurfactant is a triblock copolymer of polyethyleneoxide-polypropylene oxide-polyethylene oxide.
 12. The composition ofclaim 11, wherein the non-ionic non-fluorosurfactant is Pluronic®. 13.The composition of claim 12, wherein the concentration of the Pluronic®is in a range from about 0.1%-3.0% (weight percent).
 14. The compositionof claim 1, wherein the oil comprises a fluorous oil.
 15. Thecomposition of claim 14, wherein the fluorous oil is2-trifluoromethyl-3-ethoxydodeca-fluorohexane (HFE-7500) or1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)pentane(HFE-7300).
 16. The composition of claim 1, wherein the intercalatingdye includes EvaGreen® dye.
 17. The composition of claim 1, wherein thenucleic acid includes a target nucleic acid, and wherein only a subsetof the droplets include a copy of the target nucleic acid.
 18. Thecomposition of claim 17, wherein the aqueous droplets further compriseprimers for amplification of the target nucleic acid.
 19. Thecomposition of claim 17, wherein the aqueous droplets further comprise athermostable polymerase.
 20. The composition of claim 19, wherein theaqueous droplets are configured to amplify the target nucleic acid whenthermally cycled.